Hybridization probes derived from the spacer region between the 16S and 23S rRNA genes for the detection of Neisseria gonorrhoeae

ABSTRACT

The invention relates to a probe consisting of at least about 15 nucleotides from the spacer region between rRNA genes of a non-viral organism, particularly prokaryotic organism and more particularly bacteria, and preferably from about 15 nucleotides to about the maximum number of nucleotides of the spacer region and more preferably from about 15 to about 100 nucleotides to be used for the detection of non-viral microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a file wrapper continuing application of U.S.Ser. No. 07/965,394 filed Dec. 17, 1992, now abandoned, which is anational stage application of PCT Application No. PCT/EP91/00743 filedApr. 18, 1991, claiming priority of Great Britain Application No.90401054.3 which was filed on Apr. 18, 1990.

The invention relates to nucleic acid probes derived from the spacerregion between the ribosomal ribonucleic acid (rRNA) gene, particularlybetween the 16S and 23S rRNA genes, to be used for the specificdetection of non-viral organisms in a biological sample by ahybridization procedure.

Although much progress has been made in the last decade, for manymicroorganisms the diagnostic procedures currently in use are stilllaborious, nonsensitive and aspecific. Many of these pitfalls can beovercome by using nucleic acid probes. These nucleic acid probes can,for instance, be total genomic deoxyribonucleic acid (DNA), plasmids,riboprobes or synthetic oligonucleotides and these probes may target thegenomic DNA or messenger or stable RNA species present in biologicalsamples. Although not necessary, the use of synthetic oligonucleotidesis preferred. Oligonucleotides can be rapidly synthesized in largeamounts using chemical methods, have a long shelf-life, and are easilypurified and labeled.

For a reliable diagnosis of microorganisms using DNA-probe technologythe probes used should be highly specific (i.e. they should notcross-react with nucleic acids from other organisms) and highlysensitive (i.e. most, if not all, strains of the organism to be detectedshould react with the probe). Hence, the preferred target sequencesshould have the following characteristics:

(i) The sequence should be present in the genome of each strain of theorganism concerned.

(ii) The evolutionary diversity of the sequence should be such that, onthe one hand, there is sufficient sequence-diversity to allowdifferentiation of the species concerned from other closely relatedspecies and, on the other hand, sufficient sequence-conservation toallow the detection of all strains of the species concerned with theprobe used.

Species-specific probes have been described for a large number oforganisms. For a recent review see Tenover, Clin. Microbiol. Rev.1:82-101, 1988.

However, it is not obvious from which gene in the genome that specificprobe sequences can be derived. In probe development often largeselection procedures have to be followed to obtain fragments which atlast turn out to be specific for the organism under investigation(Korolik et al., J. Gen. Microbiol. 134:521-529, 1988; Grimont et al.,J. Clin. Microbiol. 21:431-437, 1985; Welcher et al., Nucl. Acids Res.14:10027-10044, 1986; Donegan et al., Mol. Cell. Probes 3:13-26, 1989;Beaulieu and ROY, Abstract nr D249, Abstracts of the Annual Meeting ofthe American Society for Microbiology, 1989). Most often the function oridentity of the gene from which the specific fragment derives is notknown and the screening procedure has to be blindly repeated each timeanother specific probe is wanted. The precise identification of a genewhich meets the criteria listed above and which is ubiquitously presentwould obviate the need for time-consuming and tedious selections.

The 16S or 23S rRNA genes are quite often used for probe developmentsince sequences can easily be obtained using described methods and it isknown that variable regions exist within these highly conserved geneswhich can be used for species-specific detection. However, for certainorganisms it may not be possible to derive highly specific and sensitiveprobes from the 16S and 23S rRNA genes, for instance, because theirevolutionary nucleic acid sequence conservation is too high. Anotherconsequence of the conserved character of these genes is that thedifferentiation of two organisms is often based on one or a fewmismatches only in the target sequence which puts constraints on thestringency of the hybridization. A slight deviation from theseconditions may result in misidentification.

Therefore the characterization of a ubiquitous gene which allows thedevelopment of species-specific probes for most organisms includingthose for which it was not possible to infer specific probes from the16S and 23S rRNA genes, and which preferably have a broaderstringency-range, would be extremely advantageous.

Each cellular organism possesses ribosomal RNA cistrons since itstranscripts are essential for the function of ribosomes and thesynthesis of proteins. In general the genes are present in multiplecopies in the genome. In eubacteria the 16S rRNA gene [also called smallsubunit rRNA (srRNA)] is found at the 5' end of the rRNA cistron,followed by the 23S rRNA [also called large subunit rRNA(lrRNA)]. The 5SrRNA gene is located at the 3' end of the cistron. The 16S, 23S and 5Sgenes are separated by spacer regions in which transfer RNA (tRNA) genesand signal sequences involved in post-transcriptional processing may befound. At first the rRNA cistron is transcribed as one precursor RNAmolecule. This primary transcript is further processed by endo- andexoribonucleases to its mature products. As a consequence, spacer regionsequences are not exclusively present in the genome of the organism butalso in precursor RNA molecules and processing products. The structureand processing of eubacterial rRNA cistrons is discussed in detail inthe following reference: Gegenheimer and Apirion, Microbiol. Rev.45:502-541, 1981.

The situation in nuclear genomes of eukaryotes somewhat differs in thata 5.8S RNA gene is located between the srRNA and lrRNA and 5S rRNA genesare arranged in separate long tandem arrays (Perry, Annu. Rev. Biochem.45:605-629, 1976; Long and Dawid, Annu. Rev. Biochem. 49:727-764,1980.). However, rRNA cistrons in the mitochondria or chloroplasts ofeukaryotic organisms are prokaryotic in nature (Borst and Grivell,Nature 290:443-444, 1981).

The nucleic acid sequence of the spacer region of only a very limitednumber of eukaryotic or prokaryotic organisms is available from theliterature (e.g. Young et al., J. Biol. Chem. 254:3264-3271, 1979; andMartens et al., System. Appl. Microbiol. 9:224-230, 1987.). From thesedata no reliable estimation of the nucleic acid sequence conservationcan be made and consequently nothing can be concluded concerning thesuitability of the spacer region for the selection of specific probes.

More precisely, concerning prokaryotes, hybridization probes derivedfrom the spacer region between the 16S and 23S rRNA genes for thedetection of microorganisms in a biological sample have not yet beendescribed. Neither are they known for the corresponding spacer regionbetween the small and large subunit rRNA genes of eukaryotes.

As far as eukaryotes are concerned, the use of a cloned fragment from aribosomal gene spacer has been described in a taxonomical study onLeishmania (Ramirez and Guevara, Mol. Bioch. Parasitol. 22:177-183,1987. However, the region used as well as the approach of the study areof no help to the man skilled in the art, for using a probe derived fromthe spacer region between the Small rRNA and large rRNA genes,particularly for the following reasons:

(i) the ribosomal genes spacer used by Ramirez and Guevara is not thespacer region between the srRNA and lrRNA, but refers to the sequencepresent between two adjacent rRNA cistrons; such spacers are only foundin eukaryotes between repeating units of rRNA cistrons and are notrelated to the internal spacer in between the srRNA and lrRNA genes;

(ii) the differentiation between Leishmania taxa using the gene spacerfragment is achieved by comparing restriction fragment patterns; thefragment used is not specific.

Hence, differentiation with the fragment using a simple hybridizationprotocol without resorting to Southern blot analysis is not possible.

No evidence is presented that highly specific probes can be found inthat ribosomal gene spacer.

Thus, the aim of the invention is to provide species-specific probesderived from the spacer region between rRNA genes for a particularorganism such as a bacterial species.

Another object of the invention is to provide DNA probes derived fromthe 16S-23S rRNA spacer region for the detection of Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, and Campylobacterjejuni and Campylobacter coli strains.

Still, another object of the invention is to provide DNA probes derivedfrom the 16S-23S rRNA gene spacer region for the detection of Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, and Campylobacterjejuni and Campylobacter coli strains in a biological sample by ahybridization test such as a dot-spot, strand-displacement, competition,sandwich, or reversed hybridization test. Still another object of theinvention is to provide probes and a simple method for the in vitrodiagnosis of Neisseria gonorrhoeae, Neisseria meningitidis, Branhamellacatarrhalis, Haemophilus ducreyi, Haemophilus influenzae, Borderellapertussis, Streptococcus agalactiae, Streptococcus pneumoniae, andCampylobacter jejuni and Campylobacter coli strains.

The invention relates to a probe consisting of at least about 15nucleotides of the spacer region between rRNA genes of a non-viralorganism, particularly of a prokaryotic organism, and more particularlyof a bacteria.

The invention relates more particularly to a probe consisting from about15 nucleotides to about the maximum number of nucleotides of the spacerregion, and more preferably from about 15 to about 100 nucleotides ofthe spacer region between the rRNA genes, particularly between 16S and23S rRNA genes of a non-vital organism, particularly of prokaryoticorganisms, and more particularly of bacteria.

In the following, the expression "spacer region" designates the spacerregion between rRNA genes and more particularly between the 16S and 23SrRNA genes.

The invention relates to a probe for use in a hybridization assay,liable to be obtained in the process which comprises constructing anoligonucleotide that is sufficiently complementary to hybridize to asequence of the spacer region between rRNA genes selected to be uniqueto non-viral organisms, particularly to prokaryotic organisms, moreparticularly to bacteria, which are to be detected with said sequence ofthe spacer region between rRNA genes being selected

either by

comparing the nucleotide sequence of the spacer region between the rRNAgenes of the sought organism with the nucleotide sequence of the spacerregion between the rRNA genes of the closest neighbours,

selecting a sequence of about at least 15 nucleotides, and preferablyfrom about 15 to about the maximum number of nucleotides of the spacerregion and more preferably from about 15 to about 100 nucleotides, ofthe spacer region between rRNA genes of the sought organism whichpresents at least one mismatch with the spacer region between the rRNAgenes of at least one of the closest neighbours,

or by

deleting, in the spacer region of the organism to be sought, the tRNAgenes and possibly the signal sequences, to obtain a shortened spacerregion and

determining by trial and error a specific nucleotide sequence of atleast about 15 nucleotides, and preferably from about 15 to about themaximum number of nucleotides of the spacer region and more preferablyfrom about 15 to about 100 nucleotides, from the shortened spacerregion, said sequence being able to hybridize specifically with thenucleic acids (DNA and/or RNAs) of the sought organism.

The invention relates particularly to a probe wherein the spacer regionbetween rRNA genes is the transcribed spacer region between the 16S rRNAgene and the 23S rRNA gene.

The spacer regions of several microorganisms were cloned, sequenced andcompared as will be outlined later herein. The comparison revealed thatthe nucleic acid sequence of the spacer region is of a semi-conservednature, as compared with that of rRNA genes, which are highly conserved.Hence, the spacer region might be better suited for probe developmentthan the rRNA genes itself. FIGS. 1, 2, and 10 illustrate that there isa high degree of sequence homology between highly related organisms(such as highly related strains from the same genospecies). Somewhatmore sequence variations were found between moderately related organismsas shown in FIGS. 3 and 7. A total lack of significant sequence homology(except for the tRNA sequences) could be demonstrated between distantlyrelated species, as shown in FIGS. 4 to 6.

In the Table below, homology values (in % sequence homology) of 16S rRNAsequences of different strains (16S hom) are compared with thecorresponding homology values of the spacer regions (spacer hom). Thehomology values (16S hom and spacer hom) were calculated using the PCGene software supplied by Intelligentics Inc. and Genofit SA (release6.01/Apr. 20, 1989). The total number of nucleotides compared is givenbetween parentheses. The results clearly show that the spacer region isless conserved than the 16S rRNA molecule.

    ______________________________________                                        strains compared      165       Spacer                                        strain 1    Strain 2      hom       hom                                       ______________________________________                                        N. gonorrhoeae                                                                            N. gonorrhoeae                                                                              99.9%     100%                                      NCTC 8375   ITG 4367      (1434)    (335)                                     B. pertussis                                                                              B. bronchiseptica                                                                           100%      98.1%                                     ATCC 10380  NCTC 452      (417)     (582)                                     N. gonorrhoeae                                                                            N. meningitidis                                                                             99%       93.5%                                     NCTC 8375   NCTC 10025    (1452)    (603)                                     B. catarrhalis                                                                            M. nonliquefaciens                                                                          97.9%     87.1%                                     ITG 4197    ATCC 19975    (1244)    (498)                                     B. pertussis                                                                              N. gonorrhoeae                                                                              86.3%     58.4%                                     ATCC 10380  NCTC 8375     (998)     (582)                                     B. catarrhalis                                                                            N. gonorrhoeae                                                                              83.8%     68.1%                                     ITG 4197    NCTC 8375     (1485)    (498)                                     H. ducreyi  E. coli       88.3%     67.1%                                     CIP 541                   (1498)    (346)                                     ______________________________________                                    

As a result, highly species-specific and sensitive probes could beinferred from the spacer region sequence of the relevant pathogenicspecies under study, i.e. Neisseria gonorrhoeae, Neisseria meningitidis,Branhamella catarrhalis, Haemophilus ducreyi, Haemophilus influenzae,Bordetella pertussis, Streptococcus agalactiae, Streptococcuspneumoniae, and Campylobacter jejuni and Campylobacter coli strains.Valuable probes could also be derived from the spacer region for thespecies Neisseria meningitidis and Bordetella pertussis for which highlyspecific probes could not be found in the 16S and/or 23S rRNA molecules.It is very likely that specific probes for other species than thosedescribed herein (e.g. other Campylobacter species, other Haemophilusspecies, Actinobacillus species, Bacteroides species, Chlamydia species,etc.) can be inferred from spacer region sequences as well.

The target of the probes derived from the transcribed spacer regionbetween the 16S and 23S rRNA gene is the genomic DNA and the precursorRNA molecules present in the cells to be detected. The detection of theprecursor RNA molecules is advantageous since these molecules aresingle-stranded and may be present in multiple copies. On the otherhand, DNA molecules are much more refractory to enzymatical degradationthan RNA molecules. Hence, DNA targeting is preferred when biologicalsamples cannot be processed and/or stored adequately to prevent RNAdegradation prior to hybridization.

Another particular advantage of probes derived from the 16S - 23S rRNAtranscribed spacer regions lies in target detection after enzymaticalamplification using the polymerase chain reaction (PCR). The spacerregion of many microorganisms can for instance be enzymaticallyamplified using the same primers allocated in a conserved region of the3'-end and the 5'-end of the 16S and the 23S rRNA genes respectively.Taking advantage of the highly conserved character of the rRNA genes,spacer regions of many organisms can be amplified, if preferredsimultaneously, using the same reagents and protocol and afterwards theamplified fragment can be detected using a probe which specificallytargets the spacer region of the organism of interest. An advantageousmethod for the simultaneous and specific detection of simultaneouslyamplified fragments is the reversed hybridization.

Since the spacer region is flanked by conserved sequences, the cloningand sequencing of this region with the aid of the PCR technique issimple, and the same protocol can be applied to a great variety oforganisms. Hence, the sequences of the spacer regions are obtained byenzymatical amplification of rRNA genes using conserved primersallocated in the 16S or 23S rRNA. Examples of basic primer pairs whichcan be used for the amplification of fragments spanning the spacerregion are: ##STR1##

The amplified fragment can be cloned as such or as two sub-fragmentsafter digestion with a restriction enzyme recognizing a uniquerestriction site. A strategy for cloning PCR products in M13 has beendescribed by Medlin et al. (Gene 71:491-499, 1988).

The same strategy can be used for cloning in a plasmid vector. In thisapproach the basic primers are extended at their 5'-end with anucleotide sequence comprising an unique restriction site, enablingdirectional cloning of the fragment. After cloning in a plasmid vectorthe spacer region can be sequenced using the dideoxy chain-terminationmethod.

This approach is considerably less tedious and time-consuming than theconventional cloning procedures using genomic banks or selectedrestriction endonuclease fragments.

Although sequence information is more rapidly obtained when thesequencing reactions are performed directly on PCR fragments withoutcloning, the sequence information generated from cloned fragments ismore accurate and complete. In contrast to PCR fragments, cloned genefragments can easily be purified in large amounts, which results inclearly readable sequencing ladders. Since one mismatch in the probesequence may result in useless probes, accuracy is highly preferred overspeed when obtaining sequences.

Taking into account the ease of obtaining spacer sequences with theapproach outlined above, nucleotide sequence comparison of the spacerregion of the organism for which a probe is desired with the spacerregion of the closest neighbour is the preferred way to infer specificprobe sequences.

The closest neighbour means the taxon which is known to be most closelyrelated in terms of DNA homology and which has to be differentiated fromthe organism of interest.

Depending on the taxonomical position of the organism of interest, theclosest neighbour may be very highly related to the organism ofinterest, exhibiting more than 75% degree of binding, or may be ratherdistantly related showing no significant percentage of DNA homology. Inthe initial renaturation rate method the degree of binding values areinsignificant below about 30%; in solid phase DNA:DNA hybridizationmethods, DNA homologies become insignificant between 10 to 20% degree ofbinding.

However, when the nucleotide sequences of the closest neighbours fromwhich the organism of interest has to be differentiated are notavailable, the selection of the specific probes can be done by trial anderror. In that case, for each particular organism a specific proberegion, which may be located anywhere in the spacer region, has to bedefined experimentally. Only few areas in the spacer regions, such astRNA genes or signal sequences can, in certain instances, be excluded apriori as probe regions. However, since 16S-23S rRNA spacer regions ingeneral are small--usually not longer than 900 bp--good probe sequencescan be readily found without extensive screening.

By way of example, for a spacer region between the 16S and 23S rRNA geneof 700 bp, the "shortened" spacer region obtained by deleting the tRNAgene and the signal sequence can be of about 500 bp.

The term "a biological sample" as used herein refers to a specimen suchas a clinical sample (pus, sputum, blood, urine, etc.), an environmentalsample, bacterial colonies, contaminated or pure cultures, purifiednucleic acid, etc. in which the target sequence of interest is sought.

"rRNA gene spacer region derived" as used herein refers to the fact thatthe probes concerned hybridize with sequences located in the spacerregion between ribosomal RNA genes normally present in the genome ortranscript RNA molecules, no matter whether said probes are themselvesformed of DNA or RNA fragments, or whether they consist of clonedfragments (in the case of DNA) or of synthetic oligonucleotides.

A hybridization probe of the invention for detecting Neisseriagonorrhoeae strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR2## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Neisseriameningitidis strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR3## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Branhamellacatarrhalis strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR4## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Haemophilus ducreyistrains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR5## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Haemophilusinfluenzae strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR6## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Bordetellapertussis strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR7## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Streptococcuspneumoniae strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR8## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

A hybridization probe of the invention for detecting Streptococcusagalactiae strains contains:

either a sequence belonging to a nucleic acid selected from thefollowing group of nucleic acids and which includes from 15 to themaximum number of nucleotides of the selected nucleic acid, ##STR9## ora variant sequence which differs from any of the preceding sequenceseither by addition to or removal from any of their respectiveextremities of one or several nucleotides,

or changing within any of said sequences of one or more nucleotides,

or both,

yet provided that in any of the above circumstances said probe stillhybridizes with the same RNA or DNA target as the correspondingunmodified sequence.

The invention also relates to hybridization probes for detectingCampylobacter jejuni and Campylobacter coli strains containing asequence from 15 to the maximum number of nucleotides derived from the16S-23S rRNA spacer sequence shown in FIG. 10, or the corresponding onewherein T is replaced by U, or its complement, or the corresponding onewherein T is replaced by U, provided that the probes, at the appropriateconditions, hybridize specifically with DNA and/or RNA fromCampylobacter jejuni and Campylobacter coli.

In the sequences given in groups NGI1, NGI2, NMI1, NMI2, NMI3, NMI4,NMI5, NMI6, BCI1, BCI2, HDI1, HII1, HII2, BPI1, SPI1, SPI2, SPI3, SAI1,SAI2, SAI3, and SAI4 the letters stand for the following nucleotides:

A: Adenylic residue

C: Cytidylic residue

G: Guanidylic residue

T: Thymidylic residue

U: Uracylic residue

Under the expression "target" is meant a sequence complementary to anyof the sequences of groups NGI1, NGI2, NMI1, NMI2, NMI3, NMI4, NMI5,NMI6, BCI1, BCI2, HDI1, HII1, HII2, BPI1, SPI1, SPI2, SPI3, SAI1, SAI2,SAI3, and SAI4 as previously defined herein.

In cases where the probe of the invention would comprise nucleic acidelongations on either side or both of said above defined sequences--e.g.nucleic acid fragments of cloning vector or linker fragments resultingfrom the cleavage of said probe out of said cloning vector--it isunderstood that such elongations should be selected such as to avoid thepossibility that they could themselves hybridize with any othercorresponding complementary nucleic acid sequence outside of the abovetarget in a DNA of any micro-organism likely to be tested by the processof this invention as later defined. Such hybridization would be of aparasitical nature and reduce the specificity of the probe. Preferredprobes consist of nucleic acid fragments formed from any of thesequences of the groups defined above, with said fragments containingfrom 15 to the maximum number of nucleotides of the relevant nucleicacid sequence.

It is understood that in the above nucleotide sequences (and in theother ones referred to hereafter), the left end of the formulae alwayscorresponds to a 5' extremity and the right end to a 3' extremity of thesequence concerned.

When reference is further made therein to a "probe of group `X`"--with`X` from NGI1, NGI2, NMI1, NMI2, NMI3, NMI4, NMI5, NMI6, BCI1, BCI2,HDI1, HII1, HII2, BPI1, SPI1, SPI2, SPI3, SAI1, SAI2, SAI3, and SAI4--itshould be understood that such probe has a sequence included in one ofthe nucleic acids belonging to that group as defined above or furtherdefined hereafter.

It is also understood that the word "nucleotide" as used herein refersindistinctly to ribonucleotides and deoxyribonucleotides and modifiednucleotides such as inosine unless otherwise specified. The expression"nucleotides" also encompasses those which further comprise modificationgroups, e.g. chemical modification groups which do not affect theirhybridization capabilities fundamentally. Such modification groups aim,for instance, at facilitating their coupling, either directly orindirectly, with suitable markers or labels for the subsequent detectionof the probes so marked or labeled particularly in their hybridizationproducts with the relevant RNA or DNA strand, e.g. that or thoseinitially contained in a biological sample together with other DNA(s)and/or RNA(s).

For instance, such modification groups are recognizable by antibodieswhich, in turn, can be recognized specifically by other antibodies,carrying a suitable enzymatic or fluorescent or chemiluminescent label.Possible labeling procedures will further be exemplified later herein.

The invention also relates to probes having any of the sequences definedabove and in which some nucleotides are different, provided that thedifferent nucleotide(s) do(es) not alter the specificity of the probesdefined above. Some probes may consist of one of the nucleic acidsbelonging to any of the groups which are set forth above or of partthereof, with said probes however including nucleotidic elongation oneither sides thereof to the extent that such elongations do not alterthe specificity of said probes with the genetic material of Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli strains.

The invention thus provides for probes which are either replicas (thosedesignated by numbers followed by "IC" or "ICR") in terms of nucleotidesequence of sequences contained in the RNAs or DNAs of most Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli with occasionally a few insignificantdifferences in nucleotide sequences or formed of sequences, thosedesignated by bare numbers or by numbers followed by "R", complementaryto sequences included in the natural DNAs or RNAs of Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli.

More particularly, it should be noted that the target sequences in theDNAs concerned consist in any of the following successive sequencespresent in most, if not all, Neisseria gonorrhoeae, Neisseriameningitidis, Branhamella catarrhalis, Haemophilus ducreyi, Haemophilusinfluenzae, Bordetella pertussis, Streptococcus agalactiae,Streptococcus pneumoniae, or Campylobacter jejuni and Campylobacter colistrains, subject to possible insignificant natural differences from onestrain to another, whereby such natural differences are not likely toaffect the hybridization specificity of the probes of this inventionwith such targets:

In the case of Neisseria gonorrhoeae

GCGAAGTAGA ATAACGACGC ATCG (SEQ ID NO: 2) GTTTGCTTAC TTAGTCAACGGGTAGGTAAA CGAA (SEQ ID NO: 6)

In the case of Neisseria meningitidis CAGGGCGACG TCACACTTGA CC (SEQ IDNO: 10) GACGTCACAC TTGACCAAGA AC (SEQ ID NO: 14) GCACAGTAGA TAGCTATAACGAACGC (SEQ ID NO: 18) TGCACAGTAG ATAGCAATAT CGAACGCA (SEQ ID NO: 22)AATGGAACAGAATCCATTCAGGGCGACGTCACACTTGACCAGAACAAAA (SEQ ID NO: 26)GTCTGATGTT CTAGTCAACG GAATGTTAGG CAAA (SEQ ID NO: 30)

In the case of Branhamella catarrhalis CTTTGGTAAG ATGTTTAA (SEQ ID NO:34) TCCACCAAGC AAGTTTAAAC ATCAA (SEQ ID NO: 38)

In the case of Haemophilus ducreyi CAATATGCCT CGCGCATAAT AA (SEQ ID NO:42)

In the case of Bordetella pertussis AAGCCTGTCC AGAGGATGGG TGTGG (SEQ IDNO: 54)

In the case of Haemophilus influenzae AAGTGCGGTC AATTTGATGC GT (SEQ IDNO: 46) CTTTAAGTTT TCACTTCAAA GT (SEQ ID NO: 50)

In the case of Streptococcus pneumoniae TGCATTACTT GGTGATCTCT CAC (SEQID NO: 58) AAGACCAATG CGCAGTTCCT (SEQ ID NO: 62) TTCTGACCTT TCAGTCATAAACTC (SEQ ID NO: 66)

In the case of Streptococcus agalactiae AACAATTTGA ACCTTTCGAT T (SEQ IDNO: 70) AAGACGCAAA TGGCAGGTTT CC (SEQ ID NO: 74) TTCTACAATC TATTTCTAGATCGTGGA (SEQ ID NO: 78) AACCTAGTTT CTTTAAAACT AGA (SEQ ID NO: 82)

The probes according to the invention can be formed by cloning ofrecombinant plasmids containing inserts including the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the invention can also be synthesized chemically, forinstance by the conventional phospho-triester method.

The invention also relates to a process for detecting Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli strains in a biological sample, whereinsaid process comprises contacting said biological sample in which thenucleic acids (DNAs and RNAs) have been made accessible tohybridization, if need be under suitable denaturation conditions, with aprobe of the invention under conditions enabling hybridization betweenthe probe and complementary nucleic acids of the strains, which may bepresent in the sample, and detecting the hybrids possibly formed.

The process of the invention enables to discriminate Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli from most other organism such as yeast,fungi, protozoa, other bacterial strains, and/or human cells which areliable to the be present in the sample in which the organisms ofinterest are sought. The process relates to the detection of Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli strains being directly in the sample orafter the strain has been cultured.

The detection of a hybrid can be interpreted as meaning that aninfection due to Neisseria gonorrhoeae, Neisseria meningitidis,Branhamella catarrhalis, Haemophilus ducreyi, Haemophilus influenzae,Bordetella pertussis, Streptococcus agalactiae, and Streptococcuspneumoniae was present in the biological sample, when any of the probesof groups NGI1, NGI2, NMI1, NMI2, NMI3, NMI4, NMI5, NMI6, BCI1, BCI2,MDI1, HII1, HII2, BPI1, SPI1, SPI2, SPI3, SAI1, SAI2, SAI3, and SAI4 isbeing used, respectively.

According to an advantageous embodiment of the invention, in the processfor detecting Neisseria gonorrhoeae, Neisseria meningitidis, Branhamellacatarrhalis, Haemophilus ducreyi, Haemophilus influenzae, Bordetellapertussis, Streptococcus agalactiae, Streptococcus pneumoniae, orCampylobacter jejuni and Campylobacter coli strains, the probes used arethe ones hybridizing both with DNA globally and RNA of the Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, Bordetella pertussis,Streptococcus agalactiae, Streptococcus pneumoniae, or Campylobacterjejuni and Campylobacter coli strains, which may be present in thebiological sample.

The hybridization conditions can be monitored relying upon severalparameters, e.g. hybridization temperature, the nature and concentrationof the components of the media, and the temperature under which thehybrids formed are washed.

The hybridization and wash temperature is limited in upper value,according to the probe (its nucleic acid composition, kind and length)and the maximum hybridization or wash temperature of the probesdescribed herein is about 30° C. to 58° C. At higher temperaturesduplexing competes with the dissociation (or denaturation) of the hybridformed between the probe and the target.

A preferred hybridization medium contains about 3×SSC (1×SSC=0.15M NaCl,0.015M sodium citrate, pH 7.0), about 25 mM of phosphate buffer pH 7.1,and 20% deionized formamide, 0.02% Ficoll, 0.02% bovine serum albumin,0.02% polyvinylpyrrolidone and about 0.1 mg/ml sheared denatured salmonsperm DNA.

A preferred wash medium contains about 3×SSC, 25 mM phosphate buffer pH7.1 and 20% deionized formamide. Other hybridization or wash media canbe used as well.

However, when modifications are introduced, be it either in the probesor in the media, the temperatures at which the probes can be used toobtain the required specificity should be changed according to knownrelationships, such as those described in the following reference: B. D.HAMES and S. J. HIGGINS, (eds.). Nucleic acid hybridization. A practicalapproach, IRL Press, Oxford, U.K., 1985.

In this respect it should also be noted that, in general, DNA:DNAhybrids are less stable then RNA:DNA or RNA:RNA hybrids. Depending onthe nature of the hybrid to be detected, the hybridization conditionsshould be adapted accordingly to achieve specific detection.

The process for detecting Neisseria gonorrhoeae, Neisseria meningitidis,Branhamella catarrhalis, Haemophilus ducreyi, Haemophilus influenzae,Bordetella pertussis, Streptococcus agalactiae, Streptococcuspneumoniae, or Campylobacter jejuni and Campylobacter coli strainsgenerally, according to the invention can be carried out by suitablyadjusting the hybridization temperature to a value at whichhybridization is specific. In such a case, washing under more stringentconditions is not necessary.

According to another embodiment of the process of the invention, thehybridization temperature need not necessarily be adjusted to the valueat which hybridization is specific and, in particular, can be lower thanthe temperature at which hybridization is specific, provided washing iscarried out at a temperature corresponding to the value at whichhybridization is specific.

In a process embodiment for detecting Neisseria gonorrhoeae strains (andfor distinguishing them from other bacterial taxa) with a probe of groupNGI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 50° C., the media being those defined above.

In a process embodiment for detecting Neisseria gonorrhoeae strains (andfor distinguishing them from other bacterial taxa) with a probe of groupNGI2, the hybridization temperature and/or the wash temperature issuitably adjusted to about 50° C., the media being those defined above.

In a process embodiment for detecting Neisseria meningitidis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup NMI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C., the media being those defined above.

In a process embodiment for detecting Neisseria meningitidis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup NMI2, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C., the media being those defined above.

In a process embodiment for detecting Neisseria meningitidis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup NMI3, the hybridization temperature and/or the wash temperature issuitably adjusted to about 40° C., the media being those defined above.

In a process embodiment for detecting Neisseria meningitidis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup NMI4, the hybridization temperature and/or the wash temperature issuitably adjusted to about 48° C., the media being those defined above.

In a process embodiment for detecting Neisseria meningitidis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup NMIS, the hybridization temperature and/or the wash temperature issuitably adjusted to about 58° C., the media being those defined above.

In a process embodiment for detecting Neisseria meningitidis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup NMI6, the hybridization temperature and/or the wash temperature issuitably adjusted to about 50° C., the media being those defined above.

In a process embodiment for detecting Branhamella catarrhalis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup BCI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 30° C., the media being those defined above.

In a process embodiment for detecting Branhamella catarrhalis strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup BCI2, the hybridization temperature and/or the wash temperature issuitably adjusted to about 42° C., the media being those defined above.

In a process embodiment for detecting Bordetella pertussis strains (andfor distinguishing them from other bacterial taxa) with a probe of groupBPI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 55° C., the media being those defined above.

In a process embodiment for detecting Haemophilus ducreyi strains (andfor distinguishing them from other bacterial taxa) with a probe of groupHDI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 40° C., the media being those defined above.

In a process embodiment for detecting Haemophilus influenzae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup HII1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 55° C., the media being those defined above.

In a process embodiment for detecting Haemophilus influenzae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup HII2, the hybridization temperature and/or the wash temperature issuitably adjusted to about 35° C., the media being those defined above.

In a process embodiment for detecting Streptococcus agalactiae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SAI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 35° C., the media being those defined above.

In a process embodiment for detecting Streptococcus agalactiae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SAI2, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C., the media being those defined above.

In a process embodiment for detecting Streptococcus agalactiae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SAI3, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C., the media being those defined above.

In a process embodiment for detecting Streptococcus agalactiae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SAI4, the hybridization temperature and/or the wash temperature issuitably adjusted to about 37° C., the media being those defined above.

In a process embodiment for detecting Streptococcus pneumoniae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SPI1, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C., the media being those defined above.

In a process embodiment for detecting Streptococcus pneumoniae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SPI2, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C, the media being those defined above.

In a process embodiment for detecting Streptococcus pneumoniae strains(and for distinguishing them from other bacterial taxa) with a probe ofgroup SPI3, the hybridization temperature and/or the wash temperature issuitably adjusted to about 45° C, the media being those defined above.

The invention further relates to a kit for detecting specificallyNeisseria meningitidis strains containing:

a probe specific for Neisseria meningitidis i.e. a probe of group NMI1,NMI2, NMI3, NMI4, NMI5 or NMI6;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Neisseria meningitidis to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyNeisseria gonorrhoeae strains containing:

a probe specific for Neisseria gonorrhoeae i.e. a probe of group NGI1 orNGI2;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Neisseria gonorrhoeae to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyBranhamella catarrhalis strains containing:

at least one probe selected among any of those that are specific forBranhamella catarrhalis as above defined, i.e. a probe of group BCI1 orBCI2;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Branhamella catarrhalis to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyHaemophilus ducreyi strains containing:

at least one probe selected among any of those that are specific forHaemophilus ducreyi as above defined, i.e. a probe of group HDI1;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Haemophilus ducreyi to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyBorderella pertussis strains containing:

at least one probe selected among any of those that are specific forBordetella pertussis as above defined, i.e. a probe of group BPI1;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Bordetella pertussis to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyHaemophilus influenzae strains containing:

at least one probe selected among any of those that are specific forHaemophilus influenzae as above defined, i.e. a probe of group HII1 orHII2;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Haemophilus influenzae to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyStreptococcus agalactiae strains containing:

at least one probe selected among any of those that are specific forStreptococcus agalactiae as above defined, i.e. a probe of group SAI1,SAI2, SAI3, or SAI4;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Streptococcus agalactiae to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyStreptococcus pneumoniae strains containing:

at least one probe selected among any of those that are specific forStreptococcus pneumoniae as above defined, i.e. a probe of group SPI1,SPI2 or SPI3;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Streptococcus pneumoniae to be carried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The invention further relates to a kit for detecting specificallyCampylobacter jejuni and Campylobacter coli strains containing:

at least one probe selected among any of those that are specific forCampylobacter jejuni and Campylobacter coli as above defined;

the buffer or components necessary for producing the buffer enabling anhybridization reaction between these probes and only the DNAs and/orRNAs of a strain of Campylobacter jejuni and Campylobacter coli to becarried out,

the means for detecting the hybrids resulting from the proceedinghybridization, when appropriate.

The probes of the invention can be used in a sandwich hybridizationsystem which enhances the specificity of a nucleic acid probe-basedassay. The principle and the use of sandwich hybridizations in a nucleicacid probe-based assay have been already described (e.g.: DUNN andHASSEL, Cell, 12: 23-36; 1977; RANKI et al., Gene, 21: 77-85; 1983).Although direct hybridization assays have favorable kinetics, sandwichhybridizations are advantageous with respect to a higher signal-to-noiseratio. Moreover, sandwich hybridizations can enhance the specificity ofa nucleic acid probe based assay.

If properly designed, a sandwich hybridization assay indeed maximizesthe specificity of a nucleic acid probe-based test when using two probesrecognizing two different nucleic acid stretches of one and the sameorganism. The only demands which must be met are that both probes (i)hybridize to the same nucleic acid molecule of the target organism and(ii) do not hybridize to the same non-target organisms.

For two given probes I and II, the sandwich hybridization system can bedescribed as follows:

Probe n° I hybridizes to nucleic acid from organisms A and B (not withC);

Probe n° II hybridizes to nucleic acid from organisms A and C (not withB).

Since it is absolutely required that both probes hybridize to the targetnucleic acid, a detectable signal will be generated only if the nucleicacid from organism A is present in the sample. It is obvious that if oneof the probes is specific for the organism to be detected, the otherprobe can be composed of any specific or non-specific sequence providedthat it hybridizes to the same target molecule than the first probe.

The probes of the invention can be used in a sandwich hybridizationassay which is specific for Neisseria gonorrhoeae, Neisseriameningitidis, Branhamella catarrhalis, Haemophilus ducreyi, Haemophilusinfluenzae, Bordetella pertussis, Streptococcus agalactiae,Streptococcus pneumoniae, or Campylobacter jejuni and Campylobacter colirespectively in combination with another, non-specific or specific,probe hybridizing to the same target molecule. In the sandwichhybridization process, the probes can be added simultaneously or not, tothe biological sample in which the target DNA or RNA is sought.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Neisseria gonorrhoeae strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Neisseria gonorrhoeae,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Neisseria gonorrhoeae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Neisseria meningitidis strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Neisseria meningitidis,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Neisseria meningitidis to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Branhamella catarrhalis strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Branhamella catarrhalis,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Branhamella catarrhalis to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Haemophilus ducreyi strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Haemophilus ducreyi,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Haemophilus ducreyi to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Haemophilus influenzae strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Haemophilus influenzae,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Haemophilus influenzae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Bordetella pertussis strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Bordetella pertussis,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Bordetella pertussis to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Streptococcus agalactiae strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Streptococcus agalactiae,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Streptococcus agalactiae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Streptococcus pneumoniae strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Streptococcus pneumoniae,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Streptococcus pneumoniae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Campylobacter jejuni strains in abiological sample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Campylobacter jejuni,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Campylobacter jejuni to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for sandwich hybridization assay,for the detection in vitro of Campylobacter coli strains in a biologicalsample, with said kit containing:

at least two probes targeting the same nucleic acid molecule and ofwhich at least one is specific for Campylobacter coli,

the buffer or components necessary for producing the buffer enablinghybridization reaction between these probes and the DNAs and/or RNAs ofa strain of Campylobacter coli to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The probes of the invention can be used also in a competitionhybridization protocol.

In a competition hybridization, the target molecule competes with thehybrid formation between a specific probe and its complement. The moretarget is present, the lower the amount of hybrid formed between theprobe and its complement. A positive signal, which indicates that thespecific target was present, is seen by a decrease in hybridizationreaction as compared with a system to which no target was added. In aparticular embodiment, the specific oligonucleotide probe, convenientlylabeled, is hybridized with the target molecule. Next, the mixture istransferred to a recipient (e.g. a microtiter dish yell) in which aoligonucleotide complementary to the specific probe is fixed and thehybridization is continued. After washing, the hybrids between thecomplementary oligonucleotide and the probe are measured, preferablyquantitatively, according to the label used.

The oligonucleotides of the invention can be used either asamplification primers in the polymerase chain reaction technique (PCR;Mullis and Faloona, Methods in Enzymology 155:335-350, 1987) to generatespecific enzymatically amplified fragments and/or as probes to detectfragments amplified between bracketing oligonucleotide primers.

The specificity of a PCR-assisted hybridization assay can be controlledat different levels.

The amplification process or the detection process or both can bespecific. The latter case, giving the highest specificity, is preferred.Such a highly specific PCR-assisted test can be developed using theprobes of the invention.

However, in some occurrences, a non-specific amplification process,using conserved primers bracketing the detection probes of theinvention, coupled to a specific detection, might be advantageous inorder to standardize the amplification process in such a way that it canbe used for a great variety of organisms.

Amplification primers to be used in a standardized amplification processcan be found in the conserved region of the 16S and 23S rRNA geneflanking the spacer region (see Example 1).

The invention also relates to a process for the in vitro detection ofone microorganism or to the simultaneous in vitro detection of severalmicroorganisms contained in a biological sample using any of the probesof the invention and specific for the microorganism(s) to be detected,wherein the DNA and/or RNA present in the biological sample (andcomprising the target sequence) is labeled, preferably using enzymaticamplification with at least one set of primers flanking the proberegion, and wherein said biological sample is contacted with a membraneon which one or more oligonucleotide probes are dot spotted on a knownlocation, in a medium enabling specific hybridization of the amplifiedtarget sequence and the probes on the membrane and wherein the hybridsresulting from the hybridizations are detected by appropriate means.

When amplification is necessary, its aim is to amplify the targetsequence (whereby the amplification of the flanking regions of thetarget sequence also occurs) and to label only the amplified regions.

When there is enough target sequence in the biological sample,amplification is not needed.

In such a case, labeling has to be carried out, for instance, eitherchemically or by addition of specific dyes, prior to hybridization andit is to be noted that all the DNA and/or RNA present in the biologicalsample is labeled.

The invention also relates to a kit for the in vitro detection of onemicroorganism or for the simultaneous in vitro detection of severalmicroorganisms contained in a biological sample, with said kitcontaining:

at least one of the probes according to the invention and specific forthe microorganism(s) to be detected, which is dot spotted to a membrane,

the primers needed for performing enzymatic amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatic amplification and/or enabling a hybridization reaction betweenthese probes and the DNAs and/or RNAs of a microorganism ormicroorganisms which are to be detected to to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The above-mentioned process and kit involve the reversed hybridizationdot-blot assay, such as the reversed dot-blot assay described by Saikiet al. (Proc. Natl. Acad. Sci. USA, 86:6230-6234, 1989).

In this case, the target sequences can first be enzymatically amplifiedusing PCR with 5' biotinylated primers. In a second step, the amplifiedproducts are detected upon hybridization with specific oligonucleotidesimmobilized on a solid support. Several modifications of this techniquecan be envisaged such as the one described in Example 2. For example,this technique may be particularly advantageous for the simultaneous andspecific detection of a variety of microorganisms which may be presentin a particular clinical sample after PCR with universal primersflanking the spacer region and hybridization with a membrane on whichdifferent specific oligonucleotide probes for the organisms of interestare dot-spotted. Some examples of advantageous panels of specificoligonucleotide probes which can be used in a reversed hybridizationassay as described above are:

(i) sputum-panel: Moraxella (Branhamella) catarrhalis Streptococcuspneumoniae Haemophilus influenzae

(ii) CSF-panel: Neisseria meningitidis Haemophilus influenzaeStreptococcus pneumoniae

(iii) Urogenital-panel: Neisseria gonorrhoeae Haemophilus ducreyiChlamydia trachomatis Trepgnema pallidum

Evidently these panels can be extended by adding probes for otherclinically relevant micro-organisms. Also panels for other clinicalsamples, such as samples coming from peridontal pockets or bloodsamples, may be of interest.

For the PCR also nonuniversally conserved primers, for instance primerslocated in the spacer region itself, can be used and the PCR can beperformed either with one set of primers or with different sets ofprimers in the same reaction vessel.

Reversed hybridization may also be carried out without an amplificationstep. In that particular case, the nucleic acids present in the samplehave to be labeled or modified, specifically or not, for instance,chemically or by addition of specific dyes, prior to hybridization.

In most cases, the number of specific probes for the organisms ofinterest which can be derived from the spacer regions is not limited tothe probes described in this text.

For some organisms Only one or two probes are described to demonstratethe feasibility of spacer regions for the development of highly specificand sensitive probes for a variety of bacteria. The only exception isBordetella pertussis for which only one particular region (fromnucleotide 271 to 299 in the Bordetella pertussis sequence; FIG. 2, topline) of the spacer region has a specific sequence. However, from thespacer region sequence of Bordetella pertussis, probes may be devisedwhich can be valuable in the simultaneous detection of highly relatedBordetella species. Probes which detect Bordetella species other thanBordetella pertussis may also be deduced from the sequences disclosed inFIG. 2. Likewise, potentially specific probes for Moraxellanonliguefaciens and Haemophilus influenzae biogroup aegyptius may beinferred from the spacer region sequence shown in FIGS. 7 and 8,respectively.

The invention also relates to a kit for the in vitro detection of one ormore Neisseria gonorrhoeae strains in a biological sample, with said kitcontaining:

at least one probe selected among any of those according to theinvention and specific for Neisseria gonorrhoeae, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Neisseria gonorrhoeaeto be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of one ormore Neisseria meningitidis strains in a biological sample, with saidkit containing:

at least one probe selected among any of those according to theinvention and specific for Neisseria meningitidis, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Neisseriameningitidis to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Haemophilus ducreyi strains in a biological sample, with saidkit containing:

at least one probe selected among any of those according to theinvention and specific for Haemophilus ducreyi, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Haemophilus ducreyito be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Branhamella catarrhalis strains in a biological sample, withsaid kit containing:

at least one probe selected among any of those according to theinvention and specific for Branhamella catarrhalis, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Branhamellacatarrhalis to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Bordetella pertussis strains in a biological sample, with saidkit containing:

at least one probe selected among any of those according to theinvention and specific for Bordetella pertussis, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or,As of a strain of Bordetella pertussis tobe carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Haemophilus influenzae strains in a biological sample, withsaid kit containing:

at least one probe selected among any of those according to theinvention and specific for Haemophilus influenzas, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Haemophilusinfluenzae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Streptococcus pneumoniae strains in a biological sample, withsaid kit containing:

at least one probe selected among any of those according to theinvention and specific for Streptococcus pneumoniae, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Streptococcuspneumoniae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Streptococcus agalactiae strains in a biological sample, withsaid kit containing:

at least one probe selected among any of those according to theinvention and specific for Streptococcus agalactiae, which is fixed to asolid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probe,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probe and the DNAs and/or RNAs of a strain of Streptococcusagalactiae to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

The invention also relates to a kit for the in vitro detection of oneore more Campylobacter jejuni and Campylobacter coli strains in abiological sample, with said kit containing:

at least one probe selected among any of those according to theinvention and specific for Campylobacter jejuni, and at least one probeselected among any of those according to the invention and specific forCampylobacter coli, which is fixed to a solid support,

the primers needed for performing enzymatical amplification of the DNAand/or RNA containing the target sequence of the above-mentioned probes,when appropriate,

the buffers or components necessary for producing the buffers enablingenzymatical amplification and/or enabling hybridization reaction betweensaid probes and the DNAs and/or RNAs of a strain of Campylobacter jejuniand Campylobacter coli to be carried out,

the means for detecting the hybrids resulting from the precedinghybridization, when appropriate.

Conditions for the Use of Probes

The probes of the invention are advantageously labeled. Any conventionallabel can be used. The probes can be labeled by means of radioactivetracers such as ³² P, ³⁵ S, ¹²⁵ I, ³ H and ¹⁴ C.

The radioactive labeling can be carried out according to anyconventional method such as terminal labeling at the 3' or 5' positionwith the use of a radiolabeled nucleotide, a polynucleotide kinase (withor without dephosphorylation by a phosphatase), a terminal transferase,or a ligase (according to the extremity to be labeled). One of theprobes of the invention can be the matrix for the synthesis of a chainconsisting of several radioactive nucleotides or of several radioactiveand nonradioactive nucleotides.

The probes of the invention can also be prepared by chemical synthesisusing one or several radioactive nucleotides. Another method forradioactive labeling is a chemical iodination of the probes of theinvention which leads to the binding of several ¹²⁵ I atoms on theprobes.

If one of the probes of the invention is made radioactive to be used forhybridization with a nonradioactive RNA or DNA, the method of detectinghybridization will depend on the radioactive tracer used. Generally,autoradiography, liquid scintillation, gamma counting or any otherconventional method enabling one to detect an ionizing ray issued by theradioactive tracer can be used.

Nonradioactive labeling can also be used by associating the probes ofthe invention with residues having: immunological properties (e.g.antigen or hapten), a specific affinity for some reagents (e.g. ligand),properties providing a detectable enzymatic reaction (e.g. enzyme,co-enzyme, enzyme substrate or substrate taking part in an enzymaticreaction), or physical properties such as fluorescence, emission orabsorption of light at any wavelength. Antibodies which specificallydetect the hybrids formed by the probe and the target can also be used.

A nonradioactive label can be provided when chemically synthesizing aprobe of the invention, the adenosine, guanosine, cytidine, thymidineand uracyl residues thereof being liable to be coupled to other chemicalresidues enabling the detection of the probe or the hybrids formedbetween the probe and a complementary DNA or RNA fragment.

However, the nucleotidic sequence of the probe, when modified bycoupling one or more nucleotides to other chemical residues, would bethe same as the nucleotide sequence of one of the probes of theinvention.

The invention also relates to processes for detecting RNA and/or DNAwith the probes of the invention by hybridization, which have beenlabeled and can be detected as described above. In this regard,conventional methods of hybridization can be used.

For detecting cells derived from or themselves being living organisms,the RNA and/or DNA of these cells, if need be, is made accessible bypartial or total lysis of the cells using chemical or physicalprocesses, and contacted with one or several probes of the inventionwhich can be detected. This contact can be carried out on an appropriatesupport such as a nitrocellulose, cellulose, or nylon filter in a liquidmedium or in solution. This contact can take place under suboptimal,optimal conditions, or under restrictive conditions (i.e. conditionsenabling hybrid formation only if the sequences are perfectly homologouson a length of molecule). Such conditions include temperature,concentration of reactants, the presence of substances lowering theoptimal temperature of pairing of nucleic acids (e.g. formamide,dimethylsulfoxide and urea) and the presence of substances apparentlylowering the reaction volume and/or accelerating hybrid formation (e.g.dextran sulfate, polyethyleneglycol or phenol).

The elimination of a probe of the invention which has not hybridized canbe carried out by washing with a buffer solution of appropriate ionicstrength and at an appropriate temperature, with or without treatmentwith S1 nuclease or any other enzyme digesting single-strand DNA or RNAbut not digesting DNA-RNA hybrids or double-strand DNA.

In a liquid medium, the hybrids of the probe of the invention paired tothe cellular DNA or RNA fragments can be separated from the rest of theliquid medium in different ways, e.g. by chromatography overhydroxyapatite.

Then the hybridized probes are detected by means of the label on theprobe.

In order to target the chromosomal DNA fragments, after treating RNA byone or several enzymes and denaturation of DNA fragments (i.e.separation of both chains), one of the probes of the invention iscontacted with the DNA fragments under the conditions enablinghybridization, and after the time necessary to reach the end of thehybridization, the non-hybridized fragments are separated from thehybridized fragments and the label is detected as described above forthe detection of the cells.

Generally speaking, the different probes of the invention can also becontained in recombinant DNA enabling their cloning, if the presence ofa heterologous DNA is not a nuisance for the specificity of the probesin the encompassed uses.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIGS. 1 to 10, alignments of spacer regions (completely or partiallysequenced) found in various microorganisms are shown as examples.Matches and gaps are indicated by ":" and "-", respectively. For allsequences, the noncoding strand is shown in its 5'-3' orientation.

The 5' end is proximal to the 16S rRNA gene, the 3' end proximal to the23S rRNA gene.

It is to be pointed out that each nucleic acid sequence of the spacerregion between the 16S and 23S rRNA genes of each respective organismreferred to in FIGS. 1 to 10 (except the one of E. coli) is new.

In FIG. 1 the nucleic acid sequence alignment of the 16S rRNA proximalend of the spacer region between the 16S and 23S rRNA gene of Neisseriagonorrhoeae strains NCTC 8375 (top line) and ITM 4367 (SEQ ID NO: 86)(bottom line) is shown.

In FIG. 2 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Bordetella pertussis ATCC 10380 (SEQ IDNO: 87) (top line) and Bordetella bronchiseptica NCTC 452 (SEQ ID NO:88) (bottom line) is shown.

In FIG. 3 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Neisseria meningitidis NCTC 10025 (SEQID NO: 89) (top line) and Neisseria gonorrhoeae NCTC 8375 (SEQ ID NO:85) (bottom line) is shown.

In FIG. 4 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Neisseria gonorrhoeae NCTC 8375 (SEQ IDNO: 85) (top line) and Bordetella pertussis ATCC 10380 (SEQ ID NO: 87)(bottom line) is shown.

In FIG. 5 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Branhamella catarrhalis ITM 4197 (SEQ IDNO: 90) (top line) and Neisseria gonorrhoeae NCTC 8375 (SEQ ID NO: 85)(bottom line) is shown.

In FIG. 6 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Haemophilus ducreyi CIP 542 (SEQ ID NO:91) (top line) and Escherichia coli (bottom line) is shown.

In FIG. 7 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Branhamella catarrhalis ITM 4197 (SEQ IDNO: 90) (top line) and Moraxella nonliquefaciens ATCC 19975 (SEQ ID NO:92) (bottom line) is shown.

In FIG. 8 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Haemophilus influenzae (biogroupinfluenzae) NCTC 8143 (SEQ ID NO: 93) (top line) and Haemophilusinfluenzae (biogroup aegyptius) ITM 859 (SEQ ID NO: 94) (bottom line) isshown.

In FIG. 9 the nucleic acid sequence alignment of the spacer regionbetween the 16S and 23S rRNA of Streptococcus pneumoniae S90-5122 (SEQID NO: 95) (top line) and Streptococcus agalactiae U90-2817 (SEQ ID NO:96) (bottom line) is shown.

In FIG. 10 the nucleic acid sequence alignment of the 23S rRNA proximalend of the spacer region between the 16S and 23S rRNA of Campylobacterjejuni ATCC 33560 (SEQ ID NO: 97) (top line) and Campylobacter coli ATCC33559 (SEQ ID NO: 98) (bottom line) is shown.

The strains used can be obtained at the respective culture collections:

ATCC: American Type Culture Collection, Rockville, Md., U.S.A.

CIP: Collection de l'Institut Pasteur, Paris, France.

ITM: Institute of Tropical Medicine, Antwerp, Belgium.

NCTC: National Collection of Type Cultures, Central Public HealthLaboratory, London, United Kingdom.

The examples hereafter relate to the preparation of the probes of theinvention and the experimental results with respect to the specificityand sensitivity of the probes using different hybridization protocols.The following organisms of clinical relevance were selected: Neisseriagonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus ducreyi, Haemophilus influenzae, and Bordetella pertussis.

The examples illustrate that species-specific and highly sensitiveprobes could readily be found in the spacer region of all organismsstudied. Moreover, it is shown that probes could be constructed fromthis region for organisms for which no species-specific and highlysensitive probe could be found in the 16S and/or 23S rRNA molecule. Themethods used are essentially the same as described by ROSSAU et al., J.Gen. Microbiol.; 135: 1735-1745, 1989; or in the European patentapplication n° 8940/045.3 unless otherwise stated. All methods used withthe possible exception of enzymatical amplification of rRNA genefragments and reversed hybridization, are currently known to peopleskilled in the art. The enzymatical amplification of rRNA gene fragmentsspanning the 16S-23S rRNA spacer region was obtained by the polymerasechain reaction technique (PCR) performed according to therecommendations given in the "Gene Amp" kit of Perkin Elmer Cetus.Nucleotides corresponding to conserved or semi-conserved regions in therRNA molecules were used as PCR primers. The principle and protocol ofthe reversed dot-blot is described by Saiki et al. (1989).

EXAMPLE 1

Neisseria meningitidis and Neisseria gonorrhoeae both are importanthuman pathogens, responsible for meningitidis and gonorrhoeae,respectively. These organisms are very closely related and theirdifferentiation from one another and other Neisseria species iserror-prone. DNA probes specific for Neisseria meningitidis andNeisseria gonorrhoeae may aid in the correct differentiation betweenboth Neisseria species and may be used for direct detection of thesespecies in clinical samples.

A number of DNA probes have been described for the detection ofNeisseria gonorrhoeae (European Patent Application nr 0272 009 and 0337896; URDEA et al., Clin. Chem. 35: 1571-1575, 1989; TOTTEN et al., J.Infect. Dis. 148: 462-471, 1989; DONEGAN et al., Mol. Cell. Probes 3:13-26, 1989; KOLBERG et al., Mol. Cell. Probes 3: 59-72, 1989). However,some of these probes were found to cross-react with non-Neisseriagonorrhoeae strains or were not highly sensitive. None of these probeswere derived from the 16S-23S rRNA spacer region.

A DNA probe which detects Neisseria meningitidis strains has also beendescribed (KOLBERG et al., Mol. Cell. Probes 3: 59-72, 1989). Thisprobe, devised from the pilin gene of Neisseria gonorrhoeae, was neitherhighly specific nor highly sensitive for Neisseria meningitidis.

The sequence of the spacer region between the 16S and 23S rRNA gene ofthe type strains of Neisseria gonorrhoeae and Neisseria meningitidis wasdetermined using cloned material originating from a PCR fragmentspanning the spacer region. The alignment of both sequences, shown inFIG. 3, revealed several potential probe sequences.

An unexpected inserted sequence of about 60 base pairs was detected inthe spacer region of the Neisseria meningitidis strain. Oligonucleotideswith the following sequences were derived from this inserted sequence:##STR10##

Also in another area of the spacer region (from base pairs 365 to 386 inthe Neisseria meningitidis sequence in FIG. 3) a substantial degree ofmismatch was revealed between Neisseria meningitidis and Neisseriagonorrhoeae. From this area, two oligonucleotide probes (NMI3 and NGI1for the detection of Neisseria meningitidis and Neisseria gonorrhoeae,respectively) were chemically synthesized: ##STR11##

These nucleotides were 32P-labeled at their 5' ends using polynucleotidekinase or tailed at their 3' ends with digoxigenated UTP using terminaltransferase and used as hybridization probes. As target, dot-spotteddenatured genomic DNA from a large number of Neisseria meningitidis andNeisseria gonorrhoeae strains obtained from different locations andseveral strains from other bacterial taxa was used.

The hybridization-mixture was either 3×SSC, 25 mM potassium phosphatebuffer, pH 7, deionized formamide (20%, v/v), Ficoll (0.02%, w/v),bovine serum albumin (0.02%, w/v), polyvinylpyrrolidone (0.02%, w/v) andsheared, denatured salmon sperm DNA (0.1 mg/ml) or the solution given inthe protocol sheet of the nonradioactive DNA labeling and detection kit(Boehringer Mannheim) except that 3×SSC (1×SSC is: 0.15M NaCl, 0.015Msodium citrate, pH 7.0) instead of 5×SSC was used and formamide wasadded up to 20% (v/v). The wash solution contained 3×SSC, 20% formamideand 25 mM phosphate buffer pH 7.1.

The hybridization results are summarized in the table below. Thehybridization and wash temperature for each probe is indicated betweenparenthesis. All probes tested proved to be highly specific and highlysensitive for Neisseria gonorrhoeae (probe NGI1) or Neisseriameningitidis (probes NMI1, NMI2 and NMI3).

    ______________________________________                                                   No. positives strains/No. strains tested                                        NMI1     NMI2     NMI3   NGI1                                    TAXON        (45° C.)                                                                        (45° C.)                                                                        (40° C.)                                                                      (50° C.)                         ______________________________________                                        Neisseria meningitidis                                                                     52/53    10/11    56/56   0/11                                   Neisseria sp ATCC                                                                          1/1      1/1      1/1    0/1                                     4-38-31                                                                       Neisseria gonorrhoeae                                                                       0/16    0/9       0/10  10/10                                   Neisseria polysaccharea                                                                    0/3      --       0/3    0/3                                     Neissenia lactamica                                                                         0/10    --        0/10   0/10                                   Neisseria cinerea                                                                          0/4      --       0/4    2/4                                     Neisseria mucosa                                                                           0/3      --       0/3    0/3                                     Neisseria macacae                                                                          0/1      --       0/1    0/1                                     Neisseria flavescens                                                                       0/1      --       0/1    0/1                                     Neisseria subflava                                                                         0/2      --       0/2    0/2                                     Neisseria sicca                                                                            0/1      --       0/1    0/1                                     Neisseria elongata                                                                         0/2      --       0/2    0/2                                     Neisseria canis                                                                            0/1      --       0/1    0/1                                     Neisseria animalis                                                                         0/1      --       0/1    0/1                                     Neisseria denitrificans                                                                    0/1      --       0/1    0/1                                     Neisseria sp 0/5      --       0/4    0/3                                     CDC group M-5                                                                              0/1      --       0/1    0/1                                     CDC group EF-4a                                                                            0/1      --       0/1    0/1                                     Kingella denitrificans                                                                     0/2      --       0/1    0/1                                     Kingella kingae                                                                            0/1      --       0/1    0/1                                     Simonsiella muelleri                                                                       0/1      --       0/1    0/1                                     Simonsiella crassa                                                                         0/1      --       0/1    0/1                                     Simonsiella steedae                                                                        0/1      --       0/1    0/1                                     Simonsiella sp                                                                             0/1      --       0/1    0/1                                     Alvsiella filiformis                                                                       0/1      --       0/1    0/1                                     Eikenella corrodens                                                                        0/2      --       0/2    0/2                                     Chromobacterium                                                                            0/1      --       0/1    0/1                                     violaceum                                                                     Iodobacter fluviatile                                                                      0/1      --       0/1    0/1                                     Aguaspirilum dispar                                                                        0/1      --       0/1    0/1                                     Comamonas    0/1      --       0/1    0/1                                     testosteroni                                                                  Haemophilus  0/1      --       --     --                                      influenzae                                                                    Haemophilus ducrevi                                                                        0/1      --       0/1    0/1                                     Kingella indologenes                                                                       0/1      --       0/1    0/1                                     Moraxella lacunata                                                                         0/1      --       --     --                                      Moraxella    0/1      --       --     --                                      nonliquefaciens                                                               Moraxella catarrhalis                                                                      0/3      --       0/2    0/2                                     Moraxella cuniculi                                                                         0/1      --       --     --                                      Moraxella caviae                                                                           0/1      --       --     --                                      Moraxella ovis                                                                             0/1      --       --     --                                      Moraxella osloensis                                                                        0/1      --       --     --                                      Escherichia coli                                                                           0/1      0/1      0/1    0/1                                     ______________________________________                                    

The specificity of the detection with probes NMI3 and NGI3 was alsochecked after enzymatic amplification of the spacer regions with thefollowing amplification primers: ##STR12## located at the 3' end of the16S rRNA gene and the 5' end of the 23S rRNA gene, respectively. Onehundred nanograms of genomic DNA from a strain of Neisseria gonorrhoeae,Neisseria meningitidis, Haemophilus ducreyi, Bordetella pertussis andBranhamella catarrhalis was used in the PCR reaction. Afteramplification, 1/10 of the yield was loaded on an agarose gel,electrophoresed and blotted on a nylon membrane.

The membrane was consecutively hybridized with the probes NGI1 and NMI3.

Significant hybridization signals could only be detected in lanes whereNeisseria gonorrhoeae or Neisseria meningitidis material was presentwhen NGI1 or NMI3 was used as probe, respectively.

EXAMPLE 2

Bordetella pertussis is the causative agent of whooping cough. As aresult of repeated vaccination-campaigns, the disease has become a minorproblem in the industrialized countries. However, in third-worldcountries, Bordetella pertussis remains a leading cause of childhoodmortality.

Strains of three Bordetella species (Bordetella pertussin, Borderellaparapertussis and Bordetella bronchiseptica) are extremely highlyrelated (KLOOS et al., Int. J. Syst. Bacteriol. 31:173-176, 1981; DE LEYet al., Int. J. Syst. Bacteriol. 36:405-414, 1986) and should beconsidered as belonging to one genospecies. This genotypicalrelationship is also reflected in many other characteristics of thesebacteria, thereby making their phenotypical differentiation tedious.

Clinical signs of pertussis often are atypical and laboratory diagnosisis needed. As yet, no sensitive, specific and rapid test exists. Culturestill remains the method of choice, but recovery rates are low and theresults usually are available only 3 to 7 days after inoculation(FRIEDMAN, Clin. Microbiol. Rev. 4:365-376, 1988; HALPERIN et al., J.Clin. Microbiol. 27:752-757, 1989). A DNA probe-based assay may greatlyimprove the diagnosis of Bordetella pertussis infections.

Probes for the detection of Bordetella pertussis are described in theliterature (PARK et al., FEMS Microbiol. Lett. 52:19-24, 1988; McPHEATand McNALLY, J. Gen. Microbiol. 133:323-330, 1987 and FEMS Microbiol.Lett. 41:357-360, 1987; McLAFFERTY et al., Abstracts of the AnnualMeeting of the American Society for Microbiology C-168, 1986, and C-322,1987). The probe described by McLAFFERTY et al. (1986 and 1987) is nothighly specific. For the other probes described, the data presented areto scanty to infer the degree of specificity and sensitivity.

Part of the ribosomal RNA gene of the following strains wereenzymatically amplified and cloned in a plasmid vector: Bordetellapertussis ATCC 10380, Bordetella parapertussis NCTC 5952 (type strain),and Bordetella bronchiseptica NCTC 452 (type strain). The clonedfragments of the different species were partially sequenced using thedideoxy chain termination method and their sequences were compared. Thesequence information concerning the 16S rRNA gene which becameavailable, indicated that no species-specific probes could be devised(ROSSAU et al., unpublished). However, as shown in the alignment in FIG.2, a non-homologous area (from basepairs 271 to about 300) was found inthe spacer region between the 16S and 23S rRNA genes of the Bordetellapertussis and the Bordetella bronchiseptica strain.

The sequence of the spacer region of the Bordetella parapertussis strainwas virtually identical to the Bordetella bronchiseptica sequence(ROSSAU et al., unpublished).

From the area between nucleotide 271 and 295 in the spacer region ofBordetella pertussis a oligonucleotide probe with the following sequencewas derived: ##STR13##

The oligonucleotide probe was chemically synthesized and labeled withdigoxigenin-UTP using terminal transferase. The results obtained withdotspotted denatured genomic DNA as target are summarized in the tablebelow.

    ______________________________________                                                       Hybridization with BPI1 at 55° C.                       TAXON          No. positive strains/No. strains tested                        ______________________________________                                        Bordetella pertussis                                                                         4/4                                                            Bordetella parapertussis                                                                     0/3                                                            Bordetella bronchiseptica                                                                    0/3                                                            Alcaligenes denitrificans                                                                    0/1                                                            Alcaligenes paradoxus                                                                        0/1                                                            Oligella ureolytica                                                                          0/1                                                            Oligella urethralis                                                                          0/1                                                            Taylorella equigenitalis                                                                     0/1                                                            Pseudomonas cepacia                                                                          0/1                                                            Pseudomonas solanacearum                                                                     0/1                                                            Comamonas testosteroni                                                                       0/1                                                            Neisseria meningitidis                                                                       0/1                                                            Branhamella catarrhalis                                                                      0/1                                                            Haemophilus influenzae                                                                       0/1                                                            ______________________________________                                    

Under the conditions used, the probe BPI1 proved to be 100% specific and100% sensitive for Bordetella pertussis.

The hybridization mixture was as described in the protocol sheet of thenonradioactive DNA labeling and detection kit (Boehringer Mannheim)except that 3×SSC (1×SSC is: 0.15M NaCl, 0.015M sodium citrate, pH 7.0)instead of 5×SSC was used and formamide was added up to 20% (v/v). Thewash solution contained 3×SSC, 20% formamide and 25 mM phosphate bufferpH 7.1. The hybridization and wash temperature was 55° C.

Essentially the same result as those shown in the table above wereobtained when using a reversed dot-blot assay. This assay was performedas follows:

One ng of bacterial DNA from a variety of strains obtained fromdifferent bacterial species was enzymatically amplified as recommendedby the manufacturer of the Gene-Amp kit (Perkin Elmer Cetus) except thatdigoxigenin-11-dUTP (Boehringer Mannheim) was added to the amplificationmixture to a final concentration of 40 μM. Thirty cycles (1 min/95° C.,min/50° C., 1 min/72° C.) with the primers AP16 and AP23 (see example 1)were performed in a total of 50 μl, whereafter 5 μl of each PCR mix wasadded to 1 ml of hybridization mixture (composition as defined above) inthe presence of a membrane to which 0.2 pmol, 0.02 pmol and 0.002 pmolof probe BPI1 was fixed. The hybridization proceeded for one hour at 55°C. The wash step was performed at the same temperature for 10 min. Thedetection was performed as described in the non-radioactive DNA labelingand detection kit (Boehringer Mannheim). Although a distinct band couldbe observed in all samples examined after gel electrophoresis andethidium bromide staining using the reversed dot-blot protocol, aclearly positive signal was obtained exclusively with samples in whichBordetella pertussis DNA was present.

EXAMPLE 3

Branhamella catarrhalis, also known as Moraxella catarrhalis orNeisseria catarrhalis, is a fastidious, biochemically rather inertbacterium. Recently its important pathogenic potential was recognized.

Branhamella catarrhalis seems to be frequently involved in seriousinfections of the respiratory tract (HAGER et al., Rev. Infect. Dis.9:1140-1149, 1987). The diagnosis of Branhamella catarrhalis requiresculture of the organism, which may be hampered due to overgrowth by lessfastidious micro-organisms, and a battery of phenotypical tests todistinguish this organisms from commensals, such as Neisseria species,present in the oral cavity.

In some occurrences the phenotypical test are inconclusive as to theidentity of the presumptive Branhamella catarrhalis isolate since thereonly are a limited number of tests which differentiate Branhamellacatarrhalis from phenotypical similar bacteria (RIOU and GUIBOURDENCHE,Drugs 31 [suppl.3]:1-6, 1986). The use of a DNA probe based assay mayconsiderably simplify the laboratory diagnosis of Branhamellacatarrhalis. A DNA probe for Branhamella catarrhalis derived from anunspecified DNA fragment and which cross-hybridized with DNA fromNeisseria caviae was described by BEAULIEU and ROY (Abstracts of theAnnual Meeting of the American Society for Microbiology, Abstract No.D-249, 1989).

Part of the rRNA gene of Branhamella catarrhalis ITG 4197 wasenzymatically amplified by the polymerase chain reaction technique andcloned in a plasmid vector. The fragment spanning the 16S-23S rRNAspacer region was subsequently sequenced by the dideoxy chaintermination technique. The sequence is shown in FIG. 7 (top line). Fromthe sequence data, the following oligonucleotide was selected andchemically synthesized: ##STR14##

The oligonucleotide was ³² P-labeled at its 5' end with polynucleotidekinase and used as a hybridization probe. As target, dot-spotteddenatured genomic DNA of 31 Branhamella catarrhalis strains fromdifferent locations and 19 strains of other bacterial taxa was used.

The hybridization-mixture was either 3×SSC (1×SSC is: 0.15M NaCl, 0.015Msodium citrate, pH 7.0), 25 mM potassium phosphate buffer, pH 7,deionized formamide (20%, v/v), Ficoll (0.02%, w/v), bovine serumalbumin (0.02%, w/v), polyvinylpyrrolidone (0.02%, w/v) and sheared,denatured salmon sperm DNA (0.1 mg ml-1). The wash-solution contained3×SSC, 20% formamide and 5 mM phosphate buffer pH 7.1. The hybridizationand wash temperature was 30° C.

Under the conditions used, probe BCI1 hybridized to all Branhamellacatarrhalis strains. None of the strains tested belonging to otherbacterial species gave a significant hybridization signal with theprobe.

The non-Branhamella catarrhalis strains tested are:

    ______________________________________                                        Moraxella lacunata     ATCC 17967                                             Moraxella lacunata     ATCC 17952                                             Moraxella bovis        ITM 1601                                               Moraxella nonliquefaciens                                                                            ATCC 19975                                             Neisseria cuniculi     ITM 3388                                               Neisseria ovis         NCTC 11227                                             Neisseria caviae       ATCC 14659                                             Alysiella sp.          ATCC 29468                                             Moraxella osloensis    LMG 1043                                               Moraxella osloensis    ATCC 17974                                             "Moraxella paraphenylpyruvica"                                                                       LMG 5125                                               "Moraxella camembertii"                                                                              LMG 7022                                               Psychrobacter immobiles                                                                              LMG 6784                                               Acinetobacter calcoaceticus                                                                          ATCC 23055                                             Escherichia coli       B                                                      Haemophilus influenzae NCTC 8143                                              Eikenella corrodens    NCTC 10596                                             Xanthomonas maltophilia                                                                              LMG 958                                                Xanthomonas campestris LMG 568                                                ______________________________________                                    

EXAMPLE 4

Haemophilus ducreyi, the causative agent of chancroid, is a fastidiousGram-negative bacterium. The culture of this organism is both difficultand insensitive; yet it still is the method of choice for the diagnosisof Haemophilus ducreyi infections. The use of highly specific probes mayobviate the culture and increase the sensitivity of the diagnosis.Cloned DNA probes for Haemophilus ducreyi showing weak cross-reactivitywith other Haemophilus and Pasteurella species, and targeting genescoding for proteins were described by PARSONS et al. (J. Clin.Microbiol. 27:1441-1445, 1989).

Part of the rRNA gene of the type strain of Haemophilus ducreyi CIP 542was enzymatically amplified by the polymerase chain reaction and clonedin a plasmid vector.

The sequence of the spacer region between the 16S and 23S rRNA gene wasobtained by the dideoxy chain termination technique. From the nucleicacid sequence, the following oligonucleotide was selected and chemicallysynthesized: ##STR15##

The oligonucleotide was ³² P-labeled at its 5' ends or tailed at its 3'ends with digoxigenated UTP using terminal transferase and used as ahybridization probe.

As target, dot-spotted denatured genomic DNA of 41 Haemophilus ducreyistrains from different locations and several strains of other bacterialtaxa was used. The oligonucleotide probe hybridized exclusively to allHaemophilus ducreyi strains tested.

The hybridization-mixture was either 3×SSC, 25 mM potassium phosphatebuffer, pH 7, deionized formamide (20%, v/v), Ficoll (0.02%, w/v),bovine serum albumin (0.02%, w/v), polyvinylpyrrolidone (0.02%, w/v) andsheared, denatured salmon sperm DNA (0.1 mg ml-1) or the solution givenin the protocol sheet of the nonradioactive DNA labeling and detectionkit (Boehringer Mannheim) except that 3×SSC (1×SSC is: 0.15M NaCl,0.015M sodium citrate, pH 7.0) instead of 5×SSC was used and formamidewas added up to 20% (v/v). The wash solution contained 3×SSC, 20%formamide and 25 mM phosphate buffer pH 7.1. The hybridization and washtemperature was 40° C.

The non-Haemophilus ducreyi strains tested were:

Escherichia coli MC 1061

Escherichia coli B

Actinobacillus actinomycetemcomitans NCTC 9710

Actinobacillus lignieresii NCTC 4189

Haemophilus aphrophilus NCTC 5906

Haemophilus influenzae NCTC 8143

Histophilus ovis HIM 896-7

Pasteurella multocida NCTC 10322

Branhamella catarrhalis ITM 4197

Comamonas testosteroni ATCC 17407

Oligella urethralis LMG 6227

Neisseria gonorrhoeae ITM 4437

Campylobacter jejuni CCUG 11284

Acinetobacter calcoaceticus ATCC 23055

Unidentified strain ITM 3565

EXAMPLE 5

The Gram-negative bacterial species Haemophilus influenzae can besubdivided within two biogroups: influenzae and aegyptius (Casin et al.,Ann. Inst. Pasteur/Microbiol. 137B:155-163, 1986). Organisms of theinfluenzae biogroup are important respiratory tract pathogens and alsothe cause of meningitis and otitis in children. Biogroup aegyptiusisolates are the causative agent of bacterial conjunctivitis in hotclimates and seem to be associated with Brazilian Purpuric Fever(Brenner et al., J. Clin. Microbiol. 26:1524-1534, 1988). A rapiddetection of typable and non-typable Haemophilus influenzae strains canbe achieved with nucleic acid probes.

DNA probes for this species have been described in the literature(Terpstra et al., Scand. J. Infect. Dis. 19:641-646, 1987; Malouin etal. J. Clin. Microbiol. 26:2132-2138, 1988). None of these probes havebeen derived from the 16S-23S rRNA spacer region.

Part of the rRNA gene of the type strain of Haemophilus influenzae NCTC8143 was enzymatically amplified by the polymerase chain reaction andcloned in a plasmid vector.

The sequence of the spacer region between the 16S and 23S rRNA gene wasobtained by the dideoxy chain termination technique. From the nucleicacid sequence, the following oligonucleotides were selected andchemically synthesized: ##STR16##

The oligonucleotides were ³² P-labeled at their 5' ends and used ashybridization probes. As target, dot-spotted denatured genomical DNA ofbacterial taxa was used.

The hybridization results with both probes are summarized in the tablebelow. At the hybridization and wash temperatures used, probe HII1 didnot hybridize to the Haemophilus influenzae biogroup aegyptius strain.Probe HII2 hybridized to strains of both biogroups. Both probes alsohybridized at the indicated temperatures to 15 other clinical isolatesof Haemophilus influenzae biogroup influenzae obtained from theInstitute of Tropical Medicine, Antwerp, Belgium.

The hybridization mixture was 3×SSC, 25 mM potassium phosphate buffer,pH 7, deionized formamide (20%, v/v), Ficoll (0.02%, w/v), bovine serumalbumin (0.02%, w/v), polyvinylpyrrolidone (0.02%, w/v) and sheared,denatured salmon sperm DNA (0.1 mg ml-1). The wash solution contained3×SSC, 20% formamide and 25 mM phosphate buffer pH 7.1.

    ______________________________________                                                             PROBE                                                                           HII1     HII2                                          TAXON                  (50° C.)                                                                        (30° C.)                               ______________________________________                                        Haemophilus influenzae (biogroup influenzae                                                          +        +                                             NCTC 8143                                                                     Haemophilus influenzae (biogroup influenzae                                                          +        +                                             ITM 3837                                                                      Haemophilus influenzae (biogroup aegyptius                                                           -        +                                             ITM 859                                                                       Haemophilus parahaemolyticus ITM 402                                                                 -        -                                             Haemophilus parainfluenzae ITM 1094                                                                  -        -                                             Haemophilus aphrophilus NCTC 5906                                                                    -        -                                             Haemophilus ducreyi CIP 542                                                                          -        -                                             Pasteurella multocida NCTC 10322                                                                     -        -                                             Pasteurella picida ATCC 17911                                                                        -        -                                             Actinobacillus lignieresii NCTC 4189                                                                 -        -                                             Actinobacillus actinomycetemcominitans                                                               -        -                                             NCTC 9710                                                                     Histophilus ovis HIM 896-7                                                                           -        -                                             Pseudomonas cepacia ATCC 25609                                                                       -        -                                             Acinetobacter calcoaceticus ATCC 23055                                                               -        -                                             Branhamella catarrhalis LMG 5128                                                                     -        -                                             Bordetella pertussis NCTC 8189                                                                       -        -                                             Escherichia coli B     -        -                                              Neisseria meningitidis NCTC 10025                                                                   -        -                                             ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 104                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: NO                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria gonorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGATGCTGCGTTATTCTACTTCGC24                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria gonorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCGAAGTAGAATAACGACGCATCG 24                                                   (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria genorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGAAGUAGAAUAACGACGCAUCG24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria gonorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGAUGCGUCGUUAUUCUACUUCGC24                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria gonorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TTCGTTTACCTACCCGTTGACTAAGTAAGCAAAC 34                                         (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A ) ORGANISM: Neisseria gonorrhoeae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GTTTGCTTACTTAGTCAACGGGTAGGTAAACGAA34                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria gonorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GUUUGCUUACUUAGUCAACGGGUAGGUAAACGAA34                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria gonorrhoeae                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       UUGGUUUACCUACCCGUUGACUAAGUAAG CAAAC34                                         (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGTCAAGTGTGACGTCGCCCTG22                                                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CAGGGCGACGTCACACTTGACC22                                                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAGGGCGACGUCAC ACUUGACC22                                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii ) ANTI-SENSE: NO                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGUCAAGUGUGACGUCGCCCUG22                                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTTCTTGGTCAAGTGTGACGTC2 2                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       GACGTCACACTTGACCAAGAAC22                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (i ii) HYPOTHETICAL: NO                                                       (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GACGUCACACUUGACCAAGAAC22                                                      (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GUUCUUGGUCAAGUGUGACGUC 22                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                     GCGTTCGTTATAGCTATCTACTGTGC26                                                  (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          ( ii) MOLECULE TYPE: DNA (genomic)                                            (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GCACAGTAGATAGCTATAACGAACGC26                                                  (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCACAGUAGAUAGCUAUAACGAACGC 26                                                 (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Neisseria meningitidis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GCGUUCGUUAUAGCUAUCUACUGUGC26                                                  (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TGCGTTCGATATTGCTATCTACTGTGCA28                                                (2) INFORMATION FOR SEQ ID NO:22:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TGCACAGTAGATAGCAATATC GAACGCA28                                               (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                        (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      UGCACAGUAGAUAGCAAUAUCGAACGCA28                                                (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      UGCGUUCGAUAUUGCUAUCUACUGUGCA28                                                (2 ) INFORMATION FOR SEQ ID NO:25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TTTTGTT CTTGGTCAAGTGTGACGTCGCCCTGAATGGATTCTGTTCCATT50                         (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AATGGAACAGAATCCATTCAGGGCGACGTCACACTTGACCAAGAACAAAA50                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AAUGGAACAGAAUCCAUUCAGGGCGACGUCACACUUGACCAAGAACAAAA 50                         (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      UUUUGUUCUUGGUCAAGUGUGACGUCGCCCUGAAUGGAUUCUGUUCCAUU50                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TTTGCCTAACATTCCGTTGACTAGAACATCAGAC34                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GTCTGATGTTCTAGTCAACGGAATGTTAGGCAAA 34                                         (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A ) ORGANISM: Neisseria meningitidis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GUCUGAUGUUCUAGUCAACGGAAUGUUAGGCAAA34                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisserie meningitidis                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      UUUGCCUAACAUUCCGUUGACUAGAACAUCAGAC34                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      TTAAACATCTTACCAAAG 18                                                         (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CTTTGCTAAGATGTTTAA18                                                          (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CUUUGGUAAGAUGUUUAA18                                                          (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      UUAAACAUCUUACC AAAG18                                                         (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii ) ANTI-SENSE: NO                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TTGATGTTTAAACTTGCTTGGTGGA25                                                   (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TCCACCAAGCAAGTTTAAACATCAA2 5                                                  (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       UCCACCAAGCAAGUUUAAACAUCAA25                                                  (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (i ii) HYPOTHETICAL: NO                                                       (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      UUGAUGUUUAAACUUGCUUGGUGGA25                                                   (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus ducreyi                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TTATTATGCGCGAGGCATATTG 22                                                     (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus ducreyi                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                     CAATATGCCTCGCGCATAATAA22                                                      (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          ( ii) MOLECULE TYPE: RNA (genomic)                                            (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus ducreyi                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CAAUAUGCCUCGCGCAUAAUAA22                                                      (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus ducreyi                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      UUAUUAUGCGCGAGGCAUAUUG 22                                                     (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Haemophilus influenzae                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ACGCATCAAATTGACCGCACTT22                                                      (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AAGTGCGGTCAATTTGATGCGT22                                                      (2) INFORMATION FOR SEQ ID NO:47:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AAGUGCGGUCAAUUUGAUGCG U22                                                     (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                        (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      ACGCAUCAAAUUGACCGCACUU22                                                      (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      ACTTTGAAGTGAAAACTTAAAG22                                                      (2 ) INFORMATION FOR SEQ ID NO:50:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      CTTTAAG TTTTCACTTCAAAGT22                                                     (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      CUUUAAGUUUUCACUUCAAAGU22                                                      (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      ACUUUGAAGUGAAAACUUAAAG 22                                                     (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bordetella pertussis                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      CCACACCCATCCTCTGGACAGGCTT25                                                   (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bordetella pertussis                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      AAGCCTGTCCAGAGGATGGGTGTGG25                                                   (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bordetella pertussis                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      AAGCCUGUCCAGAGGAUGGGUGUGG 25                                                  (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A ) ORGANISM: Bordetella pertussis                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      CCACACCCAUCCUCUGGACAGGCUU25                                                   (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GTGAGAGATCACCAAGTAATGCA23                                                     (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      TGCATTACTTGGTGATCTCTCAC 23                                                    (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      UGCAUUACUUGGUGAUCUCUCAC23                                                     (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GUGAGAGAUCACCAAGUAAUGCA23                                                     (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      AGGAACTGCGCATT GGTCTT20                                                       (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii ) ANTI-SENSE: YES                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      AAGACCAATGCGCAGTTCCT20                                                        (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      AAGACCAAUGCGCAGUUCCU2 0                                                       (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                       AGGAACUGCGCAUUGGUCUU20                                                       (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (i ii) HYPOTHETICAL: NO                                                       (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      GAGTTTATGACTGAAAGGTCAGAA24                                                    (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      TTCTGACCTTTCAGTCATAAACTC 24                                                   (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                     UUCUGACCUUUCAGUCAUAAACUC24                                                    (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          ( ii) MOLECULE TYPE: RNA (genomic)                                            (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      GAGUUUAUGACUGAAAGGUCAGAA24                                                    (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      AATCGAAAGGTTCAAATTGTT 21                                                      (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Streptococcus agalactiae                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      AACAATTTGAACCTTTCGATT21                                                       (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      AACAAUUUGAACCUUUCGAUU21                                                       (2) INFORMATION FOR SEQ ID NO:72:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      AAUCGAAAGGUUCAAAUUGUU 21                                                      (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                        (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      GGAAACCTGCCATTTGCGTCTT22                                                      (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      AAGACGCAAATGGCAGGTTTCC22                                                      (2 ) INFORMATION FOR SEQ ID NO:75:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      AAGACGC AAAUGGCAGGUUUCC22                                                     (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: NO                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      GGAAACCUGCCAUUUGCGUCUU22                                                      (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      TCCACGATCTAGAAATAGATTGTAGAA 27                                                (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      TTCTACAATCTATTTCTAGATCGTGGA27                                                 (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      UUCUACAAUCUAUUUCUAGAUCGUGGA27                                                 (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      UCCACGAUCUAGAAAUAGAUUGUAGAA 27                                                (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A ) ORGANISM: Streptococcus agalactiae                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      TCTAGTTTTAAAGAAACTAGGTT23                                                     (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      AACCTAGTTTCTTTAAAACTAGA23                                                     (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: YES                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      AACCUAGUUUCUUUAAAACUAGA 23                                                    (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: RNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      UCUAGUUUUAAAGAAACUAGGUU23                                                     (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 603 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisserai gonorrhoeae                                           (B) STRAIN: NCTC 8375                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      AGAGAAAGAAGGGGCTTTAGGCATTCACACTTATCGGTAAACTGAAAAGATGCGGAAGAA 60               GCTTGAGTGAAGGCAAGGTTCGCTTAAGAAGGGAAACCGGGTTTGTAGCTCAGCTGGTTA120               GAGCACACGCTTGATAAGCGTGAGGTCGGAGGTTCAAGTCCTCCCAGACCCACCAAGAAC180               GGGGGCATAGCTCAGTTGGTAGAGCACCTGCTTTGCA AGCAGGGGGTCATCGGTTCGATC240              CCGTTTGCCTCCACCAAAACTTTACAAATGAAAGCAAGTTTGCTGTTTTTAGCAGCTTAT300               TTTGATTTGCGAAGTAGAATAACGACGCATCGATCTTTAACAAATTGGAAAGCCGAAATC360               AACAAACAAAG ACAATGAGTTTGTTTTGATTTTTTATTCTTTGCAAAGGATAAAAAATCT420              CTCGCAAGAGAAAAGAAAACAAACATAGTATTTGGGTGATGATTGTATCGACTTAATCCT480               GAAACACAAAAGGCAGGATTAAGACACAACAAAGCAGTAAGCTTTATCAAAGTAG GGATT540              TCAAGTTTGCTTACTTAGTCAACGGGTAGGTAAACGAAGTCAAAGAAGTTCTTGAAATGA600               TAG603                                                                        (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 603 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisserai gonorrhoeae                                           (B) STRAIN: ITM 4367                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      AGAGAAAGAAGGGGCT TTAGGCATTCACACTTATCGGTAAACTGAAAAGATGCGGAAGAA60               GCTTGAGTGAAGGCAAGGTTCGCTTAAGAAGGGAAACCGGGTTTGTAGCTCAGCTGGTTA120               GAGCACACGCTTGATAAGCGTGAGGTCGGAGGTTCAAGTCCTCCCAGACCCACCAAGAAC 180              GGGGGCATAGCTCAGTTGGTAGAGCACCTGCTTTGCAAGCAGGGGGTCATCGGTTCGATC240               CCGTTTGCCTCCACCAAAACTTTACAAATGAAAGCAAGTTTGCTGTTTTTAGCAGCTTAT300               TTTGATTTGCGAAGTAGAATAACGACGCATCGAT CTTTAACAAATTGGAAAGCCGAAATC360              AACAAACAAAGACAATGAGTTTGTTTTGATTTTTTATTCTTTGCAAAGGATAAAAAATCT420               CTCGCAAGAGAAAAGAAAACAAACATAGTATTTGGGTGATGATTGTATCGACTTAATCCT480               GAAACACAA AAGGCAGGATTAAGACACAACAAAGCAGTAAGCTTTATCAAAGTAGGGATT540              TCAAGTTTGCTTACTTAGTCAACGGGTAGGTAAACGAAGTCAAAGAAGTTCTTGAAATGA600               TAG 603                                                                       (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 582 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bordetella pertussis                                             (B) STRAIN: ATCC 10380                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      AAGAGCTTGAGTGCTCGTGTCAAGTGTCCACGCTTATCGGTTGTTGTTATATAGCTGCTG60                GATCGGTGGCTGCTGATCCGAGAGAGAAAGGTTTCGCGGGTCTGTAGCTCAGTCGGTTAG120               AGCACCGTCTTGA TAAGGCGGGGGTCGTTGGTTCGAATCCAACCAGACCCACCAAGGTTT180              CCTGAGAGGGAAATGGGGGTGTAGCTCAGCTGGGAGAGCGCCTGCTTTGCAAGCAGGATG240               TCATCGGTTCGATCCCGTTCACCTCCACCAAAGCCTGTCCAGAGGATGGGTGTGGNN NGA300              GACCAGAAGGCGAGAGAGCAACGTTAGTGCTGCGAGTCAGTGTTAAGCGTTGGGTTTTGG360               CCGACAGCTATATATGTTCTTTAACAATTTGGAAGAAGCACAACGTAAAGTGTTCGTTTA420               GTAGTCGGCGCGAGTCGATGAAGACGGATAC GGGTTGTGATTGCATGATTTTGTTCCAAG480              TCTCAAGAACTGGCTGGGCGGCCAAGCGTTTGGTCAGATGCTTTGAACTTATGAACGGCA540               CAAGCGCGAATGAACAGCACCTATAAGACTTTAGTGTTATAG582                                 (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 590 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bordetella bronchiseptica                                       (B) STRAIN: NCTC 452                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      AAGAGCTTGAGTGCTCGTGTCAAGTGTCCACGCTTATCGGTTGTTGTTATATAGCTGCTG60                GATCGGTGGCTGCTGATCCGAGAGAGAAAGGTTTCGCGGGTCTGTAGCTCAGTCGGTTAG120               AGCACCGTCTTGATAAGGCGGGGGTCGTTGGTTCGA ATCCAACCAGACCCACCAAGGTTT180              CCTGAGAGGGAAATGGGGGTGTAGCTCAGCTGGGAGAGCGCCTGCTTTGCAAGCAGGATG240               TCATCGGTTCGATCCCGTTCACCTCCACCAGAGCCCGTCTTGAAGATGGGAGCGGGTTGG300               CAGGCGAGAC CAGGAAGGCGAGAGAGCAACGTTAGTGCTGCGAGTCAGTGTTAAGCGTTG360              GGTTTTGGCCGACAGCTATATATGTTCTTTAACAATTTGGAAGAAGCACAACGTAAAGTG420               TTCGTTTAGTAGTCGACGCGAGTCGATGAAGACGGATACGGGTTGTGATTGCAT GATTTT480              GTTCCAAGTCTCAAGAACTGGCTGGGCGGCCAAGCGTTTGGTCAGATGCTTTGAACTTAT540               GAACGGCACAAGCGCGAATGAACAGCACCTATAAGACTTTAGTGTTATAG590                         (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 664 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Neisseria meningitidis                                          (B) STRAIN: NCTC 10025                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      AGAGAAAGAAGAGGC TTTAGGCATTCACACTTATCGGTAAACTGAAAAAGATGCGGAAGA60               AGCTTGAGTGAAGGCAAGATTCGCTTAAGAAGAGAATCCGGGTTTGTAGCTCAGCTGGTT120               AGAGCACACGCTTGATAAGCGTGGGGTCGGAGGTTCAAGTCCTCCCAGACCCACCAAGA A180              CGGGGGGCATAGCTCAGTTGGTAGAGCACCTGCTTTGCAAGCAGGGGGTCATCGGTTCGA240               TCCCGTTTGCCTCCACCAATACTGTACAAATCAAAACGGAAGAATGGAACAGAATCCATT300               CAGGGCGACGTCACACTTGACCAAGAACAAAAT GCTGATATAATAATCAGCTCGTTTTGA360              TTTGCACAGTAGATAGCAATATCGAACGCATCGATCTTTAACAAATTGGAAAGCCGAAAT420               CAACAAACAAAGACAAAGCGTTTGTTTTGATTTTTTATTCTTTGCAAAGGATAAAAAATC480               GCTCACAA GAGAAAAGAAAACAAACACAGTATTTGGGTGATGATTGTATCGACTTAACCC540              TGAAACACAAAAGGCAGGATTAAGACACAACAAAGCAGTAAGCTTTATCAAAGTAGGAAA600               TTCAAGTCTGATGTTCTAGTCAACGGAATGTTAGGCAAAGTCAAAGAAGTT CTTGAAATG660              ATAG664                                                                       (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 498 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                            (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Branhamella catarrhalis                                         (B) STRAIN: ITM 4197                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      ACGAAGTTATCTGATTGGCAAGAATCCACAACAAGTTGTTCTTTGGTAAGATGTTTAAAA60                ACGGGTCTATAG CTCAGTTGGTTAGAGCACCGTGTTGATAACGCGGGGGTCATAAGTTCA120              AGTCTTATTAGACCCACCATTTTGGGGCCATAGCTCAGTTGGTAGAGCGCCTGCCTTGCA180               CGCAGGAGGTCAGGAGTTCGACTCTCCTTGGCTCCACCAAGCAAGTTTAAACATCA AAGC240              ATACATAAGCAATTTAAATAAGATTTCTTATTTATGCTTTTATTTTATAAACTGACGAAG300               TTTATAACATTATTTAACAACATAGTATGAGTCTGGGTTAATTATTTAATTCCAACAAAT360               AATTAACCTGGTGTTTGTACCCAATACAAA CACCAAAAAAGTAAAGAGAACTGAATCAAG420              CGTAAACATAGGTGAATCGTTACACATTACCCATACACACCAAAGACTTCCTAGAAGTCA480               GACTACTTGGGGTTGTAT498                                                         (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 346 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus ducreyi                                             (B) STRAIN: CIP 542                                                           (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                     CCAAAATAAAGACATCACAAGTACTCACACAGATTGTTTGATTGTTTTAGACAAGTCGGA60                ATACATCTTTAAATGTTGTCCCCATCTGTCTAGAGGCCTAGGACATCGCCCTTTCACGGC120               GGTAACCGGGGTTCGAANCCCCGTGGACGCCATCT AAAGATGATTTTTATTGTCTTATGT180              TCTTTAAAAAAATAGAAACAAGCTGAAAACTGAGAGATTTTCTAAAGTAGAAAGTCTGAG240               TAATCTAAAATCTTAGCTGAACAAAAGCAGCTAAGTGTTTAGTCTAAATCATTAACCACA300               AGTATATCAA TATGCCTCGCGCATAATAAAATACTTGAGGTTGTAT346                            (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 549 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: NO                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Moraxella nonliquefaciens                                       (B) STRAIN: ATCC 19975                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      ACGAGATTATCTGATTGGCAAGAATCCACAACAAGTTGTTCTTAGTAGTGTAAGTTAAAT60                TGGGTCTATAGCTCAGTTGGTTAGAGCACCGCCTTGATAA GGCGGGGGTCATAAGTTCAA120              GTCTTATTAGACCCACCATTTTGGGGTTATAGCTCAGTTGGTAGAGCGCCTGCCTTGCAC180               GCAGGAGGTCAGGAGTTCGACTCTCCTTAACTCCACCACTTACAATAAATGAGAACTAAG240               CAATCAAATTAGAT AACATAAAATTAGATTTCTTACTTCTACTTTATGTAGATGACTTAC300              AATTAACTGATGAAGTTAATTTCAATTATTTAACAACGTATATATGAGTCTGGGTTAATT360               ATTTAATTCCAACAAATAATTAACCATTCCGTCATACTCCACATCAAGCATATAAAGT TA420              AAACTTTTAGTATTGATGATGATCGGATAAAGTAAAGAGAACTGAATCAAGCGTAAACAT480               AGGTGAATCGTTACACATTACCCATACACACCAAAGACTTCCTAGAAGTCAGACTACTTG540               GGGTTGTAT 549                                                                 (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 474 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Haemophilus influenzae                                         (B) STRAIN: NCTC 8143                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      CTGAAGACGAGAGACAGCGAGTGCTCACACAGATTGGCTGATAGTTGTAGACAAGATTAA60                AAACGAAGCGAAAGCAACGTTGAAAAATAAACGTTAAAAGATAAAAAGAAAATAGAGTAT 120              CTTTAATTGATGTCCCCATCGTCTAGAGGCCTAGGACATCGCCCTTTCACGGCGGTAACC180               GGGGTTCGAATCCCCGTGGGACGCCAATTAAAGATAACTTTATTAGATTGTCTTACTGTT240               CTTTAAAAAATTGGAAACAAGCTGAAAACAAGAGATT TTCGAGAGAAAGTCTGAGTAGGC300              AAGATAGGAAAGTGAGAGGAGGGAACTGAAAAGGGAACTCTAAAAACAAAACCTGTTTTG360               CATAAAATCTTGATTGAACAAAAGCAATCAAGTGTTTAGTTGAATGAAAATACGCATCAA420               ATTGACCGCAC TTTGAAGTGAAAACTTAAAGTGATTGAAAACATTTGAGGTGAT474                    (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 476 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        ( iii) ANTI-SENSE: NO                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Haemophilus influenzae                                          (B) STRAIN: ITM 859                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      CCCAAGACGAGAGACAGCGAGTGCTCACACAGATTGGCTGATAGTTGTAGACAAGATTAA60                AAACGAAGCGAAAGCAACGTTGAAAAATAAACGTTAAAAGA TAAAAAGAAAATAGAGTAT120              CTTTAATTGATGTCCCCATCGTCTAGAGGCCTAGGACATCGCCCTTTCACGGCGGTAACC180               GGGGTTCGAATCCCCGTGGGACGCCANNNNNNNNNNNNTTTATTAGATTGTCTTACTGTT240               CTTTAAAAAATTGGAA ACAAGCTGAAAACAAGAGATTTTCGAGAGAAAGTCTGAGTAGGC300              AAGACAGGAAAGTGAAAAGAGGGAACTGAGAAGGAAACTCTAAAAACAAACCTGTTTTGT360               AAAAAAATCTTGATTGAACAAAAGTAATCAAGTGTTTAGTTGAATTAATGAGGCTGAAAG 420              TGCAGTCAAAGTACGGTATCTATTTTATATTGAGTTTTGAAAACATTTGANNNNNN476                   (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 246 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus pneumoniae                                        (B) STRAIN: S90-5122                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      AAGGATAAGGAACTGCGCATTGGTCTTGTTTAGTCTTGAGAGGTCTTGTGGGGCCTTAGC60                TCAGCTGGGAGAGCGCCTGCT TTGCACGCAGGAGGTCAGCGGTTCGATCCCGCTAGGCTC120              CATTGGTGAGAGATCACCAAGTAATGCACATTGAAAATTGAATATCTATATCAAATAGTA180               ACAAGAAAATAAACCGAAAACGCTGTAGTATTAATAAGAGTTTATGACTGAAAGGTCAGA24 0              AAATAA246                                                                     (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                       (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus agalactiae                                        (B) STRAIN: U90-2817                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      AAGGATAAGGAAACCTGCCATTTGCGTCTTGTTTAGTTTTGAGAGGTCTTGTGGGGCCTT60                AGCTCAGCTGGGAGAGCGCCTGCTTTG CACGCAGGAGGTCAGCGGTTCGATCCCGCTAGG120              CTCCATTGAATCGAAAGGTTCAAATTGTTCATTGAAAATTGAATATCTATATCAAATTCC180               ACGATCTAGAAATAGATTGTAGAAAGTAACAAGAAAATAAACCGAAAACGCTGTGAATAT240               T TAATGAGTTTTCTAGTTTTAAAGAAACTAGGTTAATAA279                                   (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 511 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii ) HYPOTHETICAL: NO                                                       (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Campylobacter jejuni                                            (B) STRAIN: ATCC 33560                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      TAAAATCTAAAGCAAGTATATAAAGTAGATTAAATATAAAATACAAACTCTATACTTAGA60                TTTATTTTTATCTTTAACTATAAAAGAATAT ACTTTAATAAATATAAATAACATATACAT120              TATGTATTTATATTTATAATGAGATTATTTAATATATATGCTTCCTTTAGGTTTTAAACC180               TAAATGTTCTTTTTAATTATCATTGTTAAGAGTCACAAGCAAGTTTTAATAAAAACAATT240               TTACAG GACTTGTTAAAGGATAAAACCTATTTATCTTTTCTTTGGTTTAACTTATATCTT300              TTAATTATCTTTATTTCTATAATAAAGAGAATATTAGATTTAAGATTTATAAATTAAAGA360               CAAGTTTCAAACTCACAGCTTAGTTGAGACTAAATCATTTAGTTTTATAT TAAGTGTTTG420              AATGCTTTCCGTCTTAAGATAAAGAAGTCTTATCATAAAAACTTTAACAAGGAAGTGATG480               CGTTTTAGAATCAATAATAAAAGGTAAAAAA511                                            (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 511 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Campylobacter coli                                              (B) STRAIN: ATCC 33559                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      TAAAATCTAA AGCAAGTATATAAAGTAGATTAAATATAAAATACAAACTCTATACTTAGA60               TTTATTTTTATCTTTAACTATAAAAGAATATACTTTAATAAATATAAATAACATATACAT120               TATGTATTTATATTTATAATGAGATTATTTAATATATATGCTTCCTTTAGGTTT TAAACC180              TAAATGTTCTTTTTAATTATCATTGTTAAGAGTCACAAGCAAGTTTTAATAAAAACAATT240               TTACAGGACTTGTTAAAGGATAAAACCTATTTATCTTTTCTTTGGTTTAACTTATATCTT300               TTAATTATCTTTATTTCTATAATAAAGAG AATATTAGATTTAAGATTTATAAATTAAAGA360              CAAGTTTCAAACTCACAGCTTAGTTGAGACTAAATCATTTAGTTTTATATTAAGTGTTTG420               AATGCTTTCCGTCTTAAGATAAAGAAGTCTTATCATAAAAACTTTAACAAGGAAGTGATG480               CGT TTTAGAATCAATAATAAAAGGTAAAAAA511                                           (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      TGGCTCAGATTGAACGCTGGCGGC24                                                    (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     CCTTTCCCTCACGGTACTGGT21                                                       (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     TGGGTGAAGTCGTAACAAGGTA22                                                      (2) INFORMATION FOR SEQ ID NO:102:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                                     CACGTCCTTCGTCGCCT17                                                           (2) INFORMATION FOR SEQ ID NO:103:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                                     TGGGTGAAGTCGTAACAAGGTA22                                                      (2) INFORMATION FOR SEQ ID NO:104:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                                     CACGTCCTTCGTCGCCT17                                                           __________________________________________________________________________

We claim:
 1. An isolated nucleotide probe from a transcribed spacerregion between the 16S and 23S rRNA genes of a prokaryotic microorganismwherein the sequence of the probe is selected from the group consistingof: ##STR17##
 2. A process for detecting Neisseria gonorrhoeae strainsin a biological sample comprising:(a) contacting nucleic acid sequencesof a biological sample suspected of including a complementary nucleicacid sequence of N. gonorrhoeae with a probe of claim 1 at a sufficienttemperature and hybridization solution to provide formation of a hybridbetween the probe and the complementary nucleic acid sequence of N.gonorrhoeac, wherein the hybrid is formed if the probe of claim 1contacts a N. gonorrhoeae nucleic acid sequence that is complementary tothe probe of claim 1; and (b) detecting N. gonorrhoeae by detectingformation of the hybrid.
 3. A process according to claim 2, furthercomprising amplifying the N. gonorrhoeae nucleic acid sequencescomplementary to a probe for detecting at least one Neisseriagonorrhoeae strain consisting of a nucleotide sequence of a spacerregion between the 16S and 23S rRNA genes of Neisseria gonorrhoeae,wherein the nucleotide sequence is selected from the group consistingof: ##STR18## in a biological sample using at least two primers andpolymerase chain reaction, wherein the first primer has a sequence thatflanks the 5' end of the complementary nucleic acid sequence and thesecond primer has a sequence that flanks the 3' end of the complementarynucleic acid sequence.